Hepatitis C diagnostics and vaccines

ABSTRACT

The present invention provides polynucleotides derived from cDNA of novel type of hepatitis C virus named Korean type hepatitis C virus (KHCV), polypeptides encoded therein, and antibodies directed against the polypeptides; and also provide diagnostics and vaccines employing any of the above materials as active ingredient(s).

FIELD OF THE INVENTION

The present invention relates to polynucleotides derived from cDNA of anovel type of hepatitis C virus Korean type hepatitis C virus (KHCV),polypeptides encoded therein and antibodies directed against thepolypeptides; and to diagnostics and vaccines employing any of thesereagents, i.e., said polynucleotides, polypeptides and antibodies, as anactive ingredient.

BACKGROUND OF THE INVENTION

In general, virus-induced hepatitis has been known to be caused byvarious hepatitis viruses including hepatitis A virus, hepatitis Bvirus, hepatitis delta virus, hepatitis E virus, Cytomegalo virus andEsptein-Barr virus; and the genotypes of the viruses have beendiscovered since 1980, facilitating the development of diagnostics,vaccines and therapeutic agents.

Further, it has been discovered that a new type of hepatitis nicknamedas non-A non-B or C hepatitis, accounts for 80 to 90% of hepatitiscaused by blood transfusion (Lancet, 2, 838-841 (1975)); and suchpost-transfusion hepatitis frequently progresses to cirrhosis orhepatocellular carcinoma up to about 50%.

The number of hepatitis C virus (HCV) present in patient's blood isgenerally very small and the identity or specificity of the antigen andantibody systems associated with HCV has not been completely understood;and therefore, there have been many difficulties for developingtherapeutic or diagnostic agents.

Consequently, the study on HCV has attracted a great deal of attentionfrom numerous researchers (see, e.g., Alter, H. J. et al., Lancet,459-463(1978); Tabor, E. et al., Lancet, 463-466 (1978); Hollinger, F.B. et al., Intervirology, 10, 60-68(1978); Wyke, R. J. et al., Lancet,520-524(1979); Bradley, D. W. et al., J. Med. Virol., 9, 253-269(1979)).

Bradley et al., as discribed in Gastroenterology, 88, 773-779(1985),were able to determine the biochemical and biophysical characteristicsof HCV by: infecting a champanzee with the serum of a hepatitis Cpatient; obtaining quantities of serum therefrom; extracting HCV fromthe serum; and analysing and studying HCV therewith.

Thereafter, many new studies were made with the HCV viruses isolated byemploying the Bradley method for the development of agents to diagnose,prevent and/or treat hepatitis C.

Choo et al. cloned a partial cDNA fragment of HCV extracted from theserum of champanzee which had been infected with the serum of ahepatitis C patient; and proved that the protein produced by expressingthe cDNA fragment in E. coli and yeast cell was immunologically reactivewith the antibodies obtained from the serum of hepatitis C patients(Science 244, 359-362(1989).

Kuo et al. disclosed in Science, 244, 362-364(1989) that C100-3 proteinprepared by expressing a partial HCV cDNA fragment, which was identifiedby Chiron Co. in U.S.A., fused with superoxide dismutase (SOD) gene inyeast was immunoreactive with the serum of hepatitis C patients and with70% of the serum from those patients with post-transfusion hepatitis.

Further, Houghton et al. described the usefulness of HCV antigens,especially C100-3, encoded in HCV genomic sequence isolated from achampanzee contracted with hepatitis C (hereinafter, it is referred toas “American type HCV”) for the preparation of vaccines and diagnosticagents capable of detecting anti-HCV antibodies (PCT WO 89/04669; WO90/11089); and, established a diagnostic method employing enzyme immunoassay with said antigens, e.g., C100-3.

On the basis of the above invention, Ortho Diagnostic Systems Inc. ofU.S.A. developed and distributed diagnostic agents for detectinganti-HCV antibodies in 1990. However, said C100-3 antigen used as theactive ingredient for the diagnostic agents reacts only with theantibodies of patients with chronic hepatitis C, not with those ofpatients with acute hepatitis C especially during the early stage of thedisease; and, further, it often exhibits false positive results due tothe reaction of the fused protein, SOD (Shimizu, Y. K. et al., Proc.Natl. Acad. Sci. U.S.A., 87, 6441 (1990)).

On the other hand, partial HCV cDNA clones were prepared by employingthe same method as of Houghton et al. from HCV taken from the serumcollected from Japanese hepatitis C patients, including 5′-terminalregion and structural genes encoding the core protein and the envelopeprotein; and the nucleotide sequence of the cDNA clones was determinedfrom which it was discovered that the sequence is different from that ofAmerican type HCV about 10˜15%, whereby the existence of a new type,what is called as Japaness type, of HCV was proven (Kubo, Y. et al.,Nucl. Acid. Res., 17, 10367-10372(1989); Kato, N. et al., Proc. Japan.Acad., 65, 219-223(1990); Kaneko, S. et al., Lancet, 335, 976(1990);Takeuchi, K. et al., Gene, 91, 287-291(1990); Takenchi, K. et al., Nucl.Acid. Res., 18, 4626(1990); Takamizawa, A. et al., J. Virol., 65,1105-1113(1991)); and, the specificity of the antigens derived fromJapanese type HCV for preparing vaccines and diagnostic agents againstJapanese type HCV was described by Okamoto, H. et al. in Japan. J. Exp.Med., 60, 167-177(1990).

Harada et al. further reported in J. Virol. 65, 3015 (1991) that whenthe core protein encoded in 5′-terminal portion of the structural genewas used for the antigen to diagnose anti-HCV antibodies which may bepresent in samples taken from putative patients, the antibodies could bedetected 6 to 8 weeks earlier from the time of infection than the caseof using C100-3 protein.

Lesniewski et al. also disclosed in Europen Patent Publication No.725354 (1990) an improved diagnostic method using multiple antigenswhich was more sensitive and specific than the method of using C100-3antigen alone; and Wang described in EP Publiction No. 442394 (1991)another diagnostic method wherein polypeptides consisting of 15˜65 aminoacids with epitope(s) selected from 10 different HCV epitopes wereemployed as antigens for detecting anti-HCV antibodies.

The above disclosures show that HCV diagnosis can be improved byempolying a mixture of polypeptides with different epitope(s) instead ofusing only one kind of antigen.

Furthermore, envelope proteins which exist on the surface of virus inthe form of glycoproteins have been surfaced as a possible means for thedevelopment of vaccines as well as diagnostic agents. In the case offlavivirus which is very similar to HCV, it has been known that envelopeproteins and non-structural protein 1(NS 1) play an important role inthe induction of an immuno reaction of a host cell, and in bindingitself to the receptors of host cell (F. Preugschart, J. Virol., 65,4749-4758 (1991)). In addition, it has been reported that the formationof antibodies against envelope proteins is closely connected to recoveryfrom hepatitis C (Lesniewski, R. et al., p 59; Watanabe et al., p 82,The 3rd International HCV Symposium, Strasbourg, France (1991)).

Further, Houghton et al. suggested the possibility that envelope 2(E2).Protein may prove to be an important antigen for preparing hepatitis Cvaccines for the reason that said E2 protein is supposed to have a closerelationship with immunoreaction mechanism since the amino terminalregion of the E2 protein exhibits a conspicuous species heterogeneity(The 3rd International HCV Symposium, p 20, Strasbourg, France, 1991);and a comparison of the nucleotide sequences between the Japanese typeHCV genome and the American type HCV genome has revealed that, while thenucleotide sequences encoding core proteins have a homology of about91%, those encoding envelope proteins have a homology of about 74%(Takeuchi, K. et al., J. Gen. Vir., 71, 3027-3033(1990)).

SUMMARY OF THE INVENTION

As shown above, HCVs discovered in different countries may exhibitheterogeneity in various regions; and such heterogeneity may be acritical factor in deciding the effectiveness of vaccines and thesensitivity and accuracy of diagnostic agents.

Accordingly, the present invention pertains to the isolation andcharacterization of a novel type of HCV which is isolated from Koreanhepatitis C patients (KHCV) and different from the already discoveredHCVs including the American type and the Japanese type.

More specifically, the present invention provides a fully sequenced cDNAof said KHCV and partially sequenced cDNAs of several HCV varieties.Portions of the cDNA sequences derived from KHCV are useful as probes orprimers to diagnose the presence of the virus in putative samples; and,diagnostic kits and methods utilizing such nucloeotide sequences alsoconstitute further aspects of the invention.

In addition, the present invention provides polypeptides encoded in theabove cDNA which are useful as reagents in diagnostic tests and/or ascomponents of vaccines.

Said polypeptides encompass various polypeptides comprising a KHCVepitope including recombinant polypeptides such as fused polypeptideswith a non-HCV protein; and purified forms thereof.

An additional aspect of the invention pertains to a recombinantexpression vector comprising an open reading frame (ORF) of KHCV cDNAwherein said ORF is operably linked to a regulatory sequence compatiblewith a desired host organism and such vector may comprise: a nucleotidesequence encoding a non-KHCV protein for the preparation of a fusedpolypeptide of a polypeptide derived from KHCV and other type(s) ofprotein or polypeptide(s); a host cell transformed with the recombinantexpression vector; and a polypeptide produced therefrom.

A further aspect of the present invention is a method for producing apolypeptide containing a KHCV epitope comprising: culturing host cellstransformed with an expression vector containing a sequence encoding apolypeptide containing a KHCV epitope; and a polypeptide containing aKHCV epitope produced thereby.

Another aspect of the invention includes monoclonal antibody directedagainst a KHCV epitope.

A still additional aspect of the invention is directed to a hybridomacell producing such monoclonal antibody.

Still further aspects of the invention are a diagnostic agent comprisingone or more polypeptides which contain one or more KHCV epitopes as (an)active component(s) for detecting anti-KHCV antibodies in putativesamples; and a diagnostic kit comprising such agent.

Still other aspects of the invention are a diagnostic agent comprisingone or more monoclonal antibodies directed against the KHCV antigen tobe detected as (an) active component(s) for dectecting HCV antigens inputative samples; and a diagnostic kit comprising such agent.

Even further aspect of the invention is a vaccine for the treatmentand/or prevention of HCV infection comprising a polypeptide containing aKHCV epitope, and an inactivated or attenuated HCV.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more readily understood by reference to theaccompanying drawings, wherein:

FIG. 1 shows the relative positions of various KHCV cDNA clones onKHCV-LBC 1;

FIGS. 2-1 to 2-16 show the nucleotide sequcence of KHCV-LBC1 (SEQ ID NO:96) and the amino acid sequcence of the polypeptide encoded therein;

FIG. 3 shows the starting nucleotide number and the ending nucleotidenumber of each cDNA clone on KHCV-LBC1;

Position in KHCV-LBC1 Name of CDNA clone (Nucleotide Number) KHCV 426from 301 to 726 KHCV 652 from 3928 to 4563 KHCV 403 from 6649 to 7050KHCV 752 from 3208 to 3960 KHCV 675 from 4264 to 4938 KHCV 240 from 616to 855 KHCV 513 from 814 to 1326 KHCV 810 from 1201 to 2016 KHCV 798from 1945 to 2742 KHCV 932 from 6892 to 7824 KHCV 496 from 7642 to 8136KHCV 847 from 7969 to 8814 KHCV 494 from 8722 to 9216 KHCV 570 from 2686to 3300  KHCV 1774 from 4903 to 6677 KHCV 266 from 9160 to 9472 KHCV 366from 1 to 366

FIG. 4 shows the comparative analysis of the nucleotide sequences ofKHCV-LBC1 and of genomes of American type HCV and Japanese type HCV;

FIG. 5 shows the comparative analysis of the amino acid sequencesencoded in KHCV-LBC1, the American type HCV and the Japanese type HCV;

FIG. 6 shows the comparative analysis of the nucleotide sequences of5′-terminal region of KHCV-LBC1 (SEQ ID NO: 98) and genomes of theAmerican type HCV (SEQ ID NO: 99) and the Japanese type HCV (SEQ ID NO:100);

FIG. 7 shows the nucleotide sequcence of cDNA fragment NS2-LBC2 (SEQ IDNO: 97) and the amino acid sequence of the polypeptide encoded therein;

FIG. 8 shows the nucleotide sequcence of cDNA fragment NS2-LBC3 (SEQ IDNO: 101) and the amino acid sequence of the polypeptide encoded therein;

FIG. 9 shows the nucleotide sequcence of cDNA fragment NS2-LBC20 (SEQ IDNO: 102) and the amino acid sequence of the polypeptide encoded therein;

FIG. 10 shows the nucleotide sequcence of cDNA fragment NS2-LBC21 (SEQID NO: 103) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 11 shows the nucleotide sequcence of cDNA fragment NS2-LBC23 (SEQID NO: 104) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 12 shows the nucleotide sequcence of cDNA fragment NS2-LBC25 (SEQID NO: 105) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 13 shows the nucleotide sequcence of cDNA fragment NS2-LBC26 (SEQID NO: 106) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 14 shows the nucleotide sequcence of cDNA fragment NS2-LBC27 (SEQID NO: 107) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 15 shows the nucleotide sequcence of cDNA fragment NS2-LBC28 (SEQID NO: 108) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 16 shows the nucleotide sequcence of cDNA fragment NS2-LBC29 (SEQID NO: 109) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 17 shows the nucleotide sequcence of cDNA fragment NS2-LBC30 (SEQID NO: 110) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 18 shows the nucleotide sequcence of cDNA fragment NS2-LBC31 (SEQID NO: 111) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 19 shows the nucleotide sequcence of cDNA fragment NS2-LBC32 (SEQID NO: 112) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 20 shows the nucleotide sequcence of cDNA fragment NS5-LBC20 (SEQID NO: 113) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 21 shows the nucleotide sequcence of cDNA fragment NS5-LBC21 (SEQID NO: 114) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 22 shows the nucleotide sequcence of cDNA fragment NS5-LBC23 (SEQID NO: 115) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 23 shows the nucleotide sequcence of cDNA fragment NS5-LBC25 (SEQID NO: 116) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 24 shows the nucleotide sequcence of cDNA fragment NS5-LBC27 (SEQID NO: 117) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 25 shows the nucleotide sequcence of cDNA fragment NS5-LBC28 (SEQID NO: 118) and the amino acid sequence of the polypeptide encodedtherein;

FIG. 26 shows the comparative analysis of the amino acid sequences ofpolypeptides encoded in NS2 region of cDNA of KHCV variants (SEQ ID NO:97), (SEQ ID NO: 101), (SEQ ID NO: 102), (SEQ ID NO: 103), (SEQ ID NO:104), (SEQ ID NO: 105), (SEQ ID NO: 106), (SEQ ID NO: 107), (SEQ ID NO:108), (SEQ ID NO: 119), (SEQ ID NO: 120), (SEQ ID NO: 119), (SEQ ID NO:120), (SEQ ID NO: 121) and (SEQ ID NO: 122), respectively included insubtype KHCV-L1 or KHCV-L2;

FIG. 27 shows the comparative analysis of the nucleotide sequeces of NS2region of cDNA of KHCV variants (SEQ ID NO: 102), (SEQ ID NO: 104), (SEQID NO: 106), (SEQ ID NO: 112) and (SEQ ID NO: 119), respectivelyincluded in subtype KHCV-L1;

FIG. 28 shows the comparative analysis of the nucleotide sequences ofNS2 region of cDNA of KHCV variants (SEQ ID NO: 97), (SEQ ID NO: 101),(SEQ ID NO: 103), (SEQ ID NO: 105), (SEQ ID NO: 107), (SEQ ID NO: 108),(SEQ ID NO: 109), (SEQ ID NO: 110), (SEQ ID NO: 111) and (SEQ ID NO:119), respectively included in subtype KHCV-L2;

FIG. 29 shows the comparative analysis of the nucleotide sequences ofNS5 region of cDNA of KHCV variants (SEQ ID NO: 113), (SEQ ID NO: 115),(SEQ ID NO: 116), (SEQ ID NO: 117), (SEQ ID NO: 118) and (SEQ ID NO:123), respectively included in subtype KHCV-L1 and KHCV-L2,respectively;

FIG. 30 shows an expression vector constructed for the purpose ofexpressing a KHCV cDNA fragment in yeast cells;

FIGS. 31A and B show the result of SDS polyacrylamide gelelectrophoresis (SDS-PAGE) after the expression of a KHCV cDNA fragmentin yeast cells, and FIG. 31B shows the result of western blottinganalysis with the gel of FIG. 31A;

FIGS. 32A and B show the results of SDS-PAGE (FIG. 31A) and westernblotting analysis (FIG. 31B) exhibiting the production of KHCV E2N andE2C polypeptides in yeast cells;

FIG. 33 shows the nucleotide sequence of a chemically synthesizedubiquitin gene (SEQ ID NO: 124);

FIG. 34 shows the expression vector comprising trp promoter for theexpression of a KHCV cDNA fragment in E. coli cells;

FIG. 35 shows the expression vector comprising tac promoter for theexpression of a KHCV cDNA fragment in E. coli cells;

FIGS. 36 to 38 show the results of SDS-PAGE after the expression of aKHCV cDNA fragment in E. coli cells;

FIGS. 39 to 41 show the results of western blotting analyses with thegels of FIGS. 36 to 38;

FIG. 42 shows the result of SDS-PAGE after the expression of a KHCV cDNAfragment fused with an MBP gene under the control of tac promoter in E.coli cells;

FIG. 43 shows the result of western blotting analysis with the gel ofFIG. 42;

FIG. 44 shows standard curves for enzyme immuno assay (EIA) varying withthe concentration of KHCV antigen(s) used for the detection of anti-HCVantibodies in samples; and

FIG. 45 shows a standard curve for EIA with monoclonal antibodies as afunction of the concentration of KHCV antigen present in samples.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are hereby incorporated in their entirety byreference.

As used herein, the following terms shall have the following meanings:

The term “hepatitis C virus” refers to a virus causative of non-A non-Bhepatitis or hepatitis C. The terms HCV and NANBV, and the terms NANBhepatitis (NANBH) and hepatitis C are used interchangeably herein,respectively.

The term “Korean type of hepatitis C virus” or “KHCV” refers to a noveltype of HCV which is isolated from Korean hepatitis C patients; andwhose cDNA has an open reading frame of a nucleotide sequence encodingthe amino acid sequence wherein the amino acids whose numbers are 842,849 and 853 are phenylalanine, leucine and threonine, respectively; orleucine, phenylalanine and alanine, respectively.

The term “epitope” refers to an antigenic determinant of a polypeptidewhich is capable of eliciting an immune response in an immunologicallycompetent host organism and/or is capable of specifically binding itselfto a complementary antibody. An epitope of the present inventiongenerally consists of at least 6 amino acids, preferably 7 or 8 aminoacids.

The term “fragment” means a polynucleotide or polypeptide comprising asubsequence of one of the cDNAs or proteins of the invention. Suchfragments can be produced by an enzymatic cleavage of larger molecules,using restriction endonucleases for the DNA and proteases for theproteins. The fragments of the invention, however, are not limited tothe products from any particular form of enzymatic cleavage; and mayinclude subsequences, the termini of which do not correspond to anyenzymatic cleavage points. Such fragments can be made, e.g., by chemicalsynthesis, using the sequence data provided herein. Protein fragmentscan also be produced by expressing DNA fragments encoding the proteinfragments. Such protein fragments can be useful in the present inventionif they contain a sufficient number of amino acid residues to constitutean immunoreactive and/or antigenic determinant.

The term “open reading frame” refers to a region of a polynucleotidesequence where successive nucleotide triplets may be read as codonsspecifying amino acids to encode a polypeptide.

The term “expression vector” refers to a cloning vehicle designed topromote the expression of polynucleotide inserts.

The term “regulatory sequence” means a DNA sequence involved inregulating the expression of a polynucleotide sequence, which comprises,for example, promoter, ribosomal binding site, and terminator.

The term “recombinant KHCV polypeptide” refers to a polypeptide whichcontains at least a 6 amino acid sequence encoded in KHCV cDNAs of FIGS.2-1 to 2-16 and FIGS. 7 to 25 and is linked to (an) amino acid(s) otherthan that to which it is linked in the polypeptide encoded in the KHCVcDNAs.

The term “purified KHCV polypeptide” refers to a KHCV polypeptide or afragment thereof which is substantially pure and homogenous, andseparated from cellular components which naturally accompany it.Generally, a purified KHCV polypeptide comprises over about 70 to 90% ofthe polypeptide, and more preferably at least 95% of the polypeptide.

The other terms used herein have normal and conventional meanings asused in the art.

The present invention will be more specifically illustrated hereinbelow.

Cloning of KHCV cDNA

KHCV cDNA library is prepared as follows:

HCV particles are isolated from the sera of Korean patients withhepatitis C by precipitation thereof with ultra-centrifuge; the HCV RNAis extracted from the HCV particles; double stranded cDNAs aresynthesized from the HCV RNA with a random primer or oligo d(T) primerand reverse transcriptase; the cDNA fragments are cloned, either afterpropagation by employing PCR or directly to UNI-ZAPXR vector (StratageneCo. 11099 N. Torrey, Pines Road., Calif., U.S.A), after attachment ofEco RI adaptor thereto, and the vector is packaged into virus particlesto prepare cDNA library (Saiki, P. K. et al., Science, 230, 1350(1985)).

Generally, hepatitis virus particles can be isolated from the serum orthe liver of-patients or champanzees contracted with hapatitis. In thepresent invention, HCV particles are isolated from the sera of hepatitisC patients; and the total RNA of HCV is extracted from the HCV particlesprecipitated by ultracentrifuge followed by phenol extraction andethanol precipitation.

Thereafter, said HCV total RNA is used as a template for the preparationof cDNA in the reaction employing a Zap-cDNA synthesis kit (Cat. No.200400, Stratagene Co., 11099 N. Torrey Pines Rd., La Jolla, Calif.92037, USA).

Said cDNA is synthesized by the reaction of reverse transcriptase, usingthe total RNA and random primer RANPSHCV or oligo d(T) primer (SEQ IDNO: 2), wherein primer RANPSHCV (SEQ ID NO: 1)(5′-TTTTTCATGATTGGTGGTGGAACTGGACCGTCTCGAGNNNNNN-3′; N refers to A, G, Tor C) and oligo d(T) primer(5′-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAG(T)₁₈-3′) comprise 6 randomnucleotides (primer RANPSHCV) or 18 T(oligo d(T) primer) in each3′-terminal region, and a recognition site of restriction endonucleaseXho I.

For the purpose of introducing a recognition site of Eco RI(5′-GAATTC-3′) into the synthesized cDNA for the convenience of cloning,an Eco RI adaptor (SEQ ID NO: 3) (5′-CCCCCCGAATTCGGCACGAG-3′)(3′-GGGGGGCTTAAGCCGTGCTC-5′) is attached to the synthesized cDNAfragments. And, thereafter, the cDNA fragments are propagated by PCRwith primer PSHCV (SEQ ID NO: 4) (5′-TTTTCATGATTGGTGGTGGA-3′) and Eco RIprimer (the upper stand of the Eco RI adaptor); the cDNA fragments aredigested partially with restriction endonucleases Eco RI and Xho I; thedigested cDNA is ligated with UNI-ZAPXR vector, a variant of λ gt 11,digested with Eco RI and Xho I; and, the resulting DNA is packaged invitro into particles of λ phage with Gigapack II Gold Packaging Kit(Cat. No. 200214, Stratagene Co., USA) followed by amplification byinfecting the particles into E. coli cells to prepare cDNA library.

The cDNA library is plated on E. coli cells to form phage plaques, whichare, then, screened by an immunological method as described by Huynh(DNA cloning: A Practical Approach, Vol. 1, pp. 49-78, IRL Press, UK(1985)) to select the phage clones reactive with the antibody in theserum of hepatitis C patients, which are supposed to be able to producepolypeptides derived from KHCV cDNA.

On the other hand, the UNI-ZAPXR vector can be excised in E. coli toproduce a phagemid pBluescript containing KHCV cDNA fragment (Short etal., Nucl. Acid. Res., 16, 7583-7600(1988)) which is easier to treat asa normal plasmid; and, further, pBluescript can be obtained optionallyas either single-stranded or double-stranded form since it has f1replication origin as well as Col E1 origin.

Double-stranded pBluescript DNA isolated from E. coli infected withpositive plaque is digested with restriction endonucleases Eco RI andXho I to confirm the existence and the length of the KHCV cDNA fragmentinserted between the Eco RI and the Xho I recognition sites by gelelectrophoresis; and the nucleotide sequence of the cDNA fragment isdetermined by using Sanger's method (Proc. Natl. Acad. Sci. U.S.A., 74,5463(1977)).

Thereafter, new oligonucleotide probes are synthesized on the basis ofthe determined nucleotide sequence of the clone cDNA to screen the cDNAlibrary for the purpose of obtaining the remaining region of a full KHCVcDNA; and, subsequently, the new cDNA clones so obtained are again usedto screen to further obtain KHCV cDNA clones. Also, a portion of KHCVcDNA may be obtained by PCR, using the primers synthesized on the basisof the predetermined nucleotide sequence of KHCV cDNA.

The overlapping cDNA fragments may be connected to determine the fullsequence of KHCV cDNA; and an open reading frame is deduced therefrom.

A KHCV cDNA which has the full cDNA sequence so obtained is designatedas KHCV-LBC1, which was deposited with American Type Culture Collection(ATCC) on May 14, 1991, with an accession number of ATCC 75008 under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purpose of Patent Procedure.

The full nucleotide sequence of KHCV-LBC1 and the amino acid sequenceencoded therein are described in FIGS. 2-1 to 2-11; and, the position ofeach cDNA clone on KHCV-LBC1 sequence is disclosed in FIGS. 1 and 3.KHCV-LBC1 has a long open reading frame consisting of 9030 nucleotidescovering from the 343rd nucleotide (A) to the 9372 nd nucleotide (G),counting from 5′-end.

The identification number of a given amino acid is assigned, hereinafterdepending on the position of the amino acid in the polypeptide encodedin the above 9030 nucleotides in the direction from the 5′- to the3′-end.

In the 5′-terminal region of KHCV-LBC1 prepared in accordance with thepresent invention, 13 more nucleotides than the Japanes type HCV (Kato,N. et al., Proc. Natl. Acad. Sci. U.S.A. 87, 95224(1990)) are found toexist. As described in FIG. 6, in comparison with the American type HCV.There is, 1 more nucleotide discovered; and 3 nucleotides among the 22nucleotides constructing a hairpin structure of the 5′-terminal regionare determined to be different. The 5′-terminal region generally playsan important role in expressing a viral gene and regulation thereof;and, a hairpin structure consisting of 22 nucleotides is supposed to bea recognition site for replicase and core protein; and, therefore, evena minute structural difference in the region may entail a significantand material difference in its role or specificity.

Similarly, the full nucleotide sequence of KHCV-LBC1 and the amino acidsequence encoded therein are compared with those of the American typeHCV and the Japanese type HCV, with the result that: in case of theAmerican type, the nucleotide sequence of KHCV-LBC1 is homologous up tothe level of about 78.3% and the amino acid sequence encoded thereinexhibits about 84.2% homology; and, is case of the Japanese type, thenucleotide sequence has a 90.9% homology, and the amino sequence has a93% homology (see FIGS. 4 to 6). The above results clearly show thatKHCV-LBC1 is a cDNA of a new type of HCV which is distinctly differentfrom the already identified HCVs.

Preparation of Partial cDNA Fragments of KHCV Variants

KHCV RNA is extracted from said KHCV isolated from the sera of hepatitisC patients, respectively; and cDNA of each KHCV RNA is synthesized byPCR to obtain cDNA fragments corresponding to the NS2 region or NS5region. The length of each cDNA fragment so obtained is about 340 bp ofNS2 and 320 bp NS5, respectively.

The cDNA fragments are inserted into M13mp18 and M13mp19 (New EnglandBiolabs, 32 Tozer Road Beverly, Mass. 01915-5599, U.S.A) to determinetheir nucleotide sequences (see FIGS. 7 to 25). Their nucleotidesequences of the NS2 region have 91 to 94% homology (see FIGS. 27 and28); and the NS5 region exhibits 96 to 99% homology (FIG. 29) while theamino acid sequences encoded in the NS2 and the NS5 regions have ahomology of 90 to 94% and 93 to 99%, respectively (see FIG. 26).

Moreover, it is also discovered that, depending on the amino acids withthe respective numbers of 842, 849 and 853 which are encoded in the NS2region, KHCVs can be divided into two subtypes: i.e., KHCV-L1 andKHCV-L2. The cDNAs of KHCV included in KHCV-L1 encode phenylalanine,leucine and threonine as the amino acids with their respectiveidentification numbers of 842, 849 and 853; while the cDNAs included inKHCV-L2 encode leucine, phenylalanine and alanine, respectively. As asubtype KHCV-L1, there are included: KHCV-LBC1, KHCV-LBC20, KHCV-LBC23,KHCV-LBC26 and KHCV-LBC32; while KHCV-L2 subtype includes: KHCV-LBC2,KHCV-LBC3, KHCV-LBC21, KHCV-LBC25, KHCV-LBC27, KHCV-LBC28, KHCV-LBC29,KHCV-LBC30 and KHCV-LBC31.

It should be noted that the above characteristics cannot be found in thecase of the American type HCV wherein the amino acids are cysteine,phenylalamine and valine. However, the Japanese type has the samecharacteristics as KHCV-L2, i.e., the amino acids in the above positionsare leucine, phenylalanine and alanine, respectively.

The M13 phage group (M13mp18-NS2L1) which contains M13mp18 phagecomprising each of the cDNAs included in KHCV-L1, except KHCV-LBC1,i.e., KHCV-LBC20, KHCV-LBC23, KHCV-LBC26 and KHCV-LBC32 was depositedwith American Type Culture Collection on Mar. 13, 1992 with theaccession number of ATCC 75211, and, the M13 phage group (M13mp18-NS2L2)which contains M13 mp phage comprising each of the cDNAs included inKHCV-L2, i.e., KHCV-LBC2, KHCV-LBC3, KHCV-LBC21, KHCV-LBC25, KHCV-LBC27,KHCV-LBC28, KHCV-LBC29, KHCV-LBC30 and KHCV-LBC31 was deposited withATCC on the same day with the accession number of ATCC 75212.

The cDNAs of this invention may be chemically synthesized in addition tothe methods given in Examples hereof, using the nucleotide sequenceinformation provided in FIGS. 2-1 to 2-16 and FIGS. 7 to 25. Suchchemical synthesis can be carried out using a known method such as thephosphoamidite solid support method of Matteucci et al. (J. Am. Chem.Soc., 103, 3185(1981)).

Further, because of the degeneracy of the genetic code, it will beunderstood that there are many potential nucleotide sequences that couldcode for the amino acid sequence shown in FIGS. 2-1 to 2-16 and FIGS. 7to 25.

Construction of an Expression Vector and Production of Protein Thereby

Various expression systems may be used to prepare an expression vectorcontaining a KHCV cDNA fragment in accordance with the presentinvention, including a vector capable of directing production of a fusedprotein with other polypeptide than the one derived from KHCV.

For instance, such a vector system may be constructed by employing aubiquitin expression system. In yeast, ubiquitin has been known to beexcised by ubiquitinase on the exact site very next to Arg-Gly-Gly(Ozkaynak et al., Nature, 312, 663-666(1987)). Bachmair reported inScience, 234, 178-186(1986) that a foreign protein fused with ubiquitincan also be excised on the site next to Arg-Gly-Gly of ubiquitin.

Accordingly, a desired KHCV protein can be obtained by expressing afused polynucleotide of a KHCV cDNA fragment and ubiquitin gene in yeastsince the fused protein is then excised to remove ubiquitin byubiquitinase of a yeast cell, and, as a result, the KHCV protein remainsalone.

Further, if the fused polynucleotide comprising a KHCV cDNA fragment anda ubiquitin gene is expressed in E. coli, the fused protein containingubiquitin would be obtained. The ubiquitin, however, can be excised invitro by ubiquitinase; and KHCV protein free from ubiquitinase can beobtained. The fused protein per se, of course, can be used for thepurpose of the invention; and so can the KHCV protein per se as long asit retains the necessary characteristic of KHCV protein, e.g.,anti-genicity of KHCV.

The above expression system may be effectively employed where thedesired protein is unstable and can be digested easily by protease in ahost cell since the ubiquitin can protect the desired protein from theprotease attack or stabilize it.

An expression vector utilizing the ubiquitin system may be prepared byinsertion of a KHCV cDNA fragment into an expression vector whichcomprises a ubiquitin gene.

On the other hand, a fused expression vector utilizing maltose bindingprotein (MBP) system may be used as an expression vector of thisinvention. In this system, KHCV cDNA fragment is connected after mal E1gene encoding MBP; and, the fused protein of MBP and KHCV protein isproduced thereby (Guam et al., Gene, 67, 21-30(1987); Maina et al.,Gene, 74, 369-373(1988); Amann et al., Gene, 40, 183-190(1985); Duplayet al., J. Biol. Chem., 259, 10606-10613(1984)).

The above MBP expression system is convenient for the reason that thefused protein containing MBP may be easily purified by utilizing theaffinity of MBP to maltose; and that MBP has an excisable site byprotease factor Xa in C-terminal region, which enables KHCV protein tobe freed from MBP.

For the purpose of obtaining a desired KHCV protein, a compatible hostcell is transformed with an expression vector containing a KHCV cDNAfragment; and the transformed cell is cultured under a condition thatallows the expression.

A KHCV cDNA fragment to be expressed may be prepared by employing arestriction endonuclease or a nuclease with a larger fragment orKHCV-LBC1; and by carrying out PCR with primers and KHCV-LBC1 or thefragments thereof as a template. The length and nucleotide sequence ofeach primer can be determined according to the position and length ofthe KHCV cDNA fragment to be expressed; and the primer may be completelyor partially complementary to any strand of double-staranded KHCV cDNA.

Once prepared and isolated, the KHCV cDNA fragment of this invention isinserted into an appropriate expression vehicle which contains theelements necessary for transcription and translation of the insertedgene sequences. Useful cloning vehicles may consist of segments of othernon-KHCV polynucleotide including synthetic DNA sequences such asvarious known bacterial plasmids, phage DNA, combinations of plasmidswhich have been modified to employ phage DNA or other expression controlsequences, or yeast plasmids.

Selection of an appropriate host organism is affected by a number offactors as known in the art. These factors include, for example,compatibility with the chosen vector, toxicity of the proteins encodedby the recombinant plasmid, ease of recovery of the desired protein,protein characteristics, biosafety and costs. A blance of these factorsmust be considered, and it must be understood that not all hosts will beequally effective for expression of a particular recombinant DNAmolecule.

Suitable host organisms which can be used in this invention include, butare not limited to, plant, mammalian, insect cells or yeast cells andbacteria such as Escherichia coli.

The polypeptides dervied from KHCV cDNA include all the core proteins,non-structural proteins and envelope proteins and a portion thereof,which could be used for preparing diagnostic agents or vaccines in theform of a mixture thereof or alone. The polypeptides produced in a hostcell may be isolated and purified by a combined use of conventionalmethods, e.g., cell disruption, centrifugation, dialysis, salting-out,chromatography, gel filtration, electrophoresis and electroelution.

The polypeptides of this invention can also be isolated from KHCVparticles, or can be chemically synthesized by a suitable method such asexclusive solid phase synthesis, partial solid phase method, fragmentcondensation or classical solution synthesis. Solid phase synthesis asdescribed by Merrifield (J. Am. Chem. Soc., 85, 2149(1963)) ispreferred.

On the other hand, amino acid substitutions in proteins which do notsubstantially alter biological and immunological activities have beenknown to occur and have been described, e.g., by Neurath et al., in “TheProteins”, Academic Press, New York (1979), in particular in FIG. 6appearing on page 14 thereof. Most frequently observed amino acidsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, Asp/Gly, and vice versa.

Such functionally equivalent amino acid substitutions of the exemplaryembodiments of this invention are within the scope of the invention aslong as the resulting proteins retain one or more antigenic determinantsof KHCV.

In this specification, standard single-letter or three-letterabbreviations are used to represent nucleotides and amino acids. Themeanings of these abbreviations can be found in standard biochemistrytextbooks, such as Lehninger, Principles of Biochemistry, WorthPublishers Inc., New York, pp. 96, 798(1984).

Diagnostic Method of Hepatitis C Using KHCV Antigen Polypeptides for theDetection of KHCV Antibodies

The present invention also relates to a diagnostic method using adiagnostic agent containing KHCV polypeptides with one or more KHCVepitopes. The diagnostic method using KHCV polypeptide(s) is a specificand accurate for detecting KHCV antibodies in the serum of hepatitis Cpatients than any of the existing methods.

The novel diagnostic method comprises the following steps:

First, a diagnostic agent containing one or more KHCV polypeptides isadded to a solid support, e.g., well of microtiter plate to make saidKHCV antigen adsorb onto the surface of the well;

Second, a putative sample diluted with a diluent is added to theantigen-coated well where the antigen-antibody complex would be formedif there were anti-KHCV antibodies in the serum;

Third, enzyme, e.g., HRP (horseradish peroxidase) conjugated anti-humanIgG is added to the well to allow the anti-human IgG-HRP to bind theantibodies of the complex formed in the second step; and

Finally, substrates for the enzyme, e.g., O-phenylene diaminedihydrochloric acid (OPD) and hydrogen peroxide for peroxidase are addedto the well to develop a color reaction. When the putative serumcontains anti-KHCV antibodies, color appears as a result of the reactionof the enzyme with the substrates. The color reaction is stopped byaddition of diluted sulfuric acid.

The degree of color intensity can be measured with a microwell reader;and the existence of anti-HCV antibodies can be determined on the basisof the result. The solid support for the diagnositc method may be ofpolystrene bead or nitrocellulose strip.

Further, the present invention provides a hepatitis C diagnostic kitwhich comprises the necessary agents to carry out the above procedure,essentially consisting of a diagnostic agent containing KHCVpolypeptide(s) which carries one or more KHCV epitopes.

Preparation of Antibodies

The present invention provides antibodies directed againstpolypeptide(s) derived from KHCV cDNA. Briefly, appropriate animals areselected and the desired immunization protocol are followed. After anappropriate period of time, the spleens of such animals are excised andindividual spleen cells are fused, typically, with myeloma cells underappropriate selection conditions. Thereafter, the cells are clonallyseparated and the supernatant of each clone is tested for its productionof an appropriate antibody specific for the desired region of theantigen.

An animal, e.g., a mouse, may be immunized by employing a conventionalmethod such as the following:

A substantially purified antigen is injected into the mouseintramuscularly, intraperitoneally, intradermally or intravenously, morespecifically, serveral times with intervals of 14 to 21 days in a totalamount of 100 to 200 μg per mouse. A conventional adjuvant such asFreund's complete adjuvant or incomplete adjuvant may be used together,if necessary. Three days after a final injection, spleen cells of themouse are removed for fusion with mouse myeloma cells whose survivalrate is over 95% and which is in log phase.

The fusion of the cells may be carried out by employing a known methodin the art, e.g., as described by Lovborg in Monoclonal antibodies:Production & Maintenance, William Heinemann, Medical Books Ltd. (1982)The fused cells so obtained are diluted serially by employing a knownmethod as described, e.g., in Current Protocols in Immunology, WileyInterscience (1991), to detect a clone which produces the desiredantibodies.

A desired clone may be screened by using a conventional method such asenzyme immuno assay, plaque method, spot method, Ouchterlony method andradioimmunoassay as described in Hybridoma Methods & MonoclonalAntibodies, Research and Development Press, pp 30-53(1982).

The desired monoclonal antibodies may be easily obtained by one skilledin the art, using the cloned antibody-producing cell line; and, furtherpurified by employing a conventional method such as affinitychromatography.

The antibodies are useful for the purification of KHCV antigens and forthe development of an improved diagnostic method to detect KHCV antigensin putative samples.

Preparation of Diagnostic Oligonucleotide Probe and Kit

On the basis of the determined nucleotide sequence of KHCV cDNAs shownin FIGS. 2-1 to 2-11 and FIGS. 7 to 25, at least 8 nucleotidescomplementary to any of the KHCV cDNA strands may be prepared byexcision or synthetically. The oligonucleotides may be used as probesfor hybridization after labelling, e.g., with radioactive labels, or asprimers for PCR with KHCV cDNAs as a template for the detection of KHCVin serum sample.

The oligonucleotides may be either completely or partially complementaryto a KHCV cDNA strand, depending on the circumstances.

The oligonucleotides should contain at least 8 nucleotides, preferably10 to 12 nucleotides, and, more preferably, about 20 nucleotides.

Preparation of Vaccines and Administration Thereof

Inactivated or attenuated KHCV prepared by employing a known method inthe art as well as one or more of the polypeptides encoded in KHCV cDNAfragments of this invention may be formulated, along with aphysiologically acceptable carrier, into vaccines. Suitable carriersinclude, e.g., 0.01 to 0.1M phosphate buffer of neutral pH orphysiological saline solution.

Enhanced immunity against HCV can be produced by adding an adjuvant orimmunopotentiator to the vaccine, or presenting the polypeptides in alarger form, either as a cross-linked complex or conjugated to a carrierform.

Suitable adjuvants for the vaccination may include, but are not limitedto, Adjuvant 65 (containing peanut oil, mannide monooleate and aluminummonostearate); mineral gels such as aluminum hydroxide, aluminumphosphate and alum; surfactants such as hexadecylamine, octadecylamine,lysolecithin, dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl)propanediamine,methoxyhexadecyclglycerol and pluronic polyols; polyanions such aspyran, dextran sulfate, poly IC, polyacrylic acid and carbopol; peptidessuch as muramyl dipeptide, dimethylglycine and tuftsin; and oilemulsions. The proteins of the present invention may also beadministered following their incorporation into liposomes or othermicrocarriers.

The immunogenicity of the proteins of the invention, especially theirsmaller fragments, can be enhanced by cross-linking or by coupling to animmunogenic carrier molecule (i.e., a macromolecule having the propertyof independently eliciting an immunological response in a host animal,to which the proteins and protein fragments of the invention can becovalently linked). Cross-linking or conjugation to a carrier moleculemay be required because small protein fragments sometimes act as haptens(molecules which are capable of specifically binding to an antibody butincapable of eliciting antibody production, i.e., which are notimmunogenic). Conjugation of such fragments to an immunogenic carriermolecule renders the fragments immunogenic through what is commonlyknown as the “carrier effect”.

Suitable carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides, etc. One of the useful carriers is a glycosidecalled Quil A, disclosed by Morein et al. (Nature, 308, 457(1984)).Protein carrier molecules are especially preferred, including, but notlimited to, mammalian serum proteins such as keyhole limpet hemocyanin,human or bovine gammaglobulin, human, bovine or rabbit serum albumin, ormethylated or other derivatives of such proteins. Other usable proteincarriers will be apparent to those skilled in the art.

Covalent coupling to a carrier molecule can be carried out by usingvarious methods well known in the art, the exact choice of which may bedictated by, e.g., the nature of the carrier molecule used. When theimmunogenic carrier molecule is a protein, the proteins or fragments ofthe invention may be coupled to such carrier protein by water solublecarbodiimides such as dicyclohexylcarbodiimide, or glutaraldehyde.

Coupling agents such as these can also be used to cross-link theproteins and their fragments to themselves so as to obviate the use of aseparate carrier molecule. Such cross-linking among the proteins ortheir fragment aggregates can also increase immunogenicity.

Incorporation into liposomes or other microcarriers may provide theeffect of releasing the vaccines over a prolonged period of time.

The vaccine may be administered in a single dose schedule, or preferablyin a multiple dose schedule. An effective dose of the polypeptidespresent in the vaccine formulas may range from about 5 to about 200 μgdepending on the body weight of the subject to be immunized, thecapacity of the subject's immune system to produce antibodies, and thedegree of immunity desired. Initial vaccinations are preferably followedby booster vaccinations given from one to several months later. Multipleboosters may be administered.

Standard routes of administration can be used such as subcutaneous,intradermal, intramuscular or intravenous administration.

The following examples are intended to specifically examplify thepresent invention without limiting the scope of the invention; and theexperimental methods used in Examples are practiced in accordance withReference Examples given hereinbelow unless otherwise stated.

Unless otherwise specified, percentages given below for solids in solidmixtures, liquids in liquids and solids in liquids are on a wt/wt,vol/vol and wt/vol basis, respectively.

REFERENCE EXAMPLE 1 Digestion of DNA with Restriction Endonuclease

Restrction enzymes and reaction buffers were purchased from NEB (NewEngland Biolabs, Jolla, Mass., U.S.A.).

The reaction was generally carried out in a sterilized eppendorf tubewith a reaction volume ranging from 50 to 100 μl, at a temperature of37° C. for 1 to 2 hours. Thereafter, the reaction mixture washeat-treated at 65° C. for 15 minutes (or extracted with phenol andprecipitated with ethanol in the case of a heat-resistant endonuclease)to inactivate the restriction endonuclease.

10×reaction buffer for the reaction of a restriction endonuclease hasthe following composition:

10×NEB reaction buffer 1: 100 mM bis Tris propane-HCl, 100 mM MgCl₂, 10mM dithiothreitol (DTT), pH 7.0

10×NEB reaction buffer 2: 100 mM Tris-HCl, 100 mM MgCl₂, 500 mM NaCl, 10mM DTT, pH 7.0

10×NEB reaction buffer 3: 100 mM Tris-HCl, 100 mM MgCl₂, 1000 mM NaCl,10 mM DTT, pH 7.0

10×NEB reaction buffer 4: 200 mM Tris-acetate, 100 mM magnesium acetate,500 mM potassium acetate, 10 mM DTT, pH 7.0

REFERENCE EXAMPLE 2 Phenol Extraction and Ethanol Precipitation

After the completion of the enzyme reaction, the reaction mixture wasextracted with phenol for the purpose of inactivating the enzyme orrecovering the DNA in the reaction mixture, wherein phenolpreequilibrated with a buffer containing 10 mM Tris-HCl (pH 8.0) and 1mM EDTA was used. Phenol extraction was carried out by mixing equalvolumes of the sample and the phenol with vigorous shaking; centrifugingthe mixture at 15,000 rpm for 5 minutes; and transferring the aqueouslayer into a new tube. The above procedure was repeated two or threetimes.

The aqueous layer was, then, extracted with an equal volume ofchloroform (chloroform:isoamyl alcohol=24:1) and the aqueous layer wasseparated again; 0.1 volume of 3M sodium acetate and 2.5 volume ofethanol were added thereto; and, the mixture was centrifuged at 15,000rpm and 4° C. for 20 minutes after having left it at −70° C. for 30minutes or at −20° C. for 12 hours, to recover the nucleic acid.

REFERENCE EXAMPLE 3 Ligation Reaction

Ligation reaction of DNA was carried out by employing T₄ DNA ligase and10×ligation reaction buffer (0.5M Tris-HCl, 0.1M MgCl₂, 0.2M DTT, 10 mMATP, 0.5 mg/ml bovine serum albumin (BSA)) purchased from NEB. Thereaction volume was generally 20 μl, and 10 units of T₄ ligase was usedfor the ligation of cohesive ends of DNA while 100 units was used forthe ligation of blunt ended DNAs.

The reaction was carried out at 16° C. for 5 hours or at 4° C. for over14 hours; and, after the reaction was completed, the reaction mixturewas heated at 65° C. for 15 minutes to inactivate T₄ DNA ligase.

REFERENCE EXAMPLE 4 Transformation of E. coli

E. coli strains used for the following examples include E. coliHB101(ATCC 33694), E. coli W3110(ATCC 27325), E. coli JM101(ATCC 33876)and E. coli JM105(ATCC 47016). Transformation of E. coli was carried outby employing a method known in the art, e.g., as described by Maniatiset al., in Molecular Cloning: A Laboratory Manual, Cold Spring HarborPress, N.Y. (1982), or by Cohen in Proc. Natl. Acad. Sci. U.S.A., 69,2110(1972).

REFERENCE EXAMPLE 5 Transformation of Yeast

Yeast was transformed using a method described by Beggs in Nature, 275,104(1978) or described by Hinnen et al., in Proc. Natl. Acad. Sci.U.S.A., 75, 1929(1978).

REFERENCE EXAMPLE 6 Synthesis of Oligonucleotides

Oligonucleotides were synthesized by employing a DNA synthesizer(Applied Biosystems Inc., 380B, U.S.A.) of automatic solid phasephosphoamidite chemistry.

The synthesized oligonucleotides were purified by using denaturingpolyacrylamide gel (2M urea, 12% acrylamide and bis (29:1), 50 mM Tris,50 mM broic acid, 1 mM EDTA) electrophoresis and SEP-PAK (Waters Inc.,U.S.A) column chromatography; and the amount was determined by measuringO.D. at 260 nm.

REFERENCE EXAMPLE 7 Polymerase Chain Reaction (PCR)

To a mixture of 10 to 100 ng of a template DNA, 10 μl of 10×Taqpolymerase reaction buffer (10 mM Tris-HCl, 500 mM KCl, 15 mM MgCl₂,0.1% (w/v) gelatin, pH 8.3), 10 μl of a mixture of dNTP's (each of dGTP,dATP, dTTP and dCTP is 2 mM), 2 μg of each primer (generally, 2 primerswere used for a reaction, and in the case that 3 primers were used, theprimer located in the middle was used in an amount of 0.02 μg), and 0.5μl of Ampli Taq DNA polymerase (Perkin Elmer Cetus, U.S.A.) was addeddistilled water in an amount to make a total volume of 100 μl; and 50 μlof mineral oil was added thereto to protect the reaction mixture fromevaporation.

The PCR was carried out by using a thermal cycler (Perkin Elmer Cetus,U.S.A.); and the thermal cycle was programmed to repeat 25 times ormore, the cycle of: 95° C. for 1 minute→55° C. for 1 minute→72° C. for 2minutes, finally, the reaction was carried out at 72° C. for 10 minutes.

After the reaction was completed, the mixture was extracted with phenoland the PCR products were recovered by precipitation with ethanol; and,the precipitate was dissolved in 20 μl of TE buffer solution (10 mMTris-HCl, 1 mM EDTA, pH 7.5).

EXAMPLE 1 Preparation of KHCV cDNA KHCV-LBC1

(1-A): Isolation of HCV from Serum of Korean Hepatitis C Patients andExtraction of the Viral Genomic RNA Therefrom

50 ml of serum from Korean patients with chronic hepatitis diagnosed asnon-A, non-B hepatitis (ALT<60IU: The serum was supplied from the KoreaUniversity Hospital and the Catholic University Hospital in Korea) wasultracentrifuged to preciptate the HCV particles by the method proposedby Bradley, D. W. et al. in Gastroenterology, 88, 773(1985). 50 ml ofserum was 6-fold diluted with TENB buffer solution (0.05M Tris, pH 8.0,0.001M EDTA (ethylene diaminetetraacetic acid), 0.1M NaCl) andultracentrifuged at 28,000 rpm, room temperature for 6 hours usingBeckman Rotor SW28 (Beckman Inc., Model L8-80M).

Extraction of the viral genomic RNA from precipitated viral particleswas carried out by using the method proposed by Cholozynski, P. andSacchi, N. in Anal. Biochem., 162, pp 156-159 (1987). The precipitatedviral particles were suspended in 8 ml of RNA extraction solution (4Mguanidine thiocyanate, 24 mM Na-citrate, pH 7.0, 0.5% sarcosyl, 0.1M2-mercaptoethanol). 0.8 ml of 2M sodium acetate (pH 4.0), 8 ml of phenol(BRL Inc., U.S.A.; saturated with distilled water) and 1.6 ml ofchloroform-isoamyl alcohol (49:1, v/v) were added thereto and theresulting mixture was then centrifuged at 12,000×g, 4° C. for 15minutes. The supernatant was poured into a new test tube; and a samevolume of isopropanol and glycogen (2 μg/ml supernatant) as carrier wasadded thereto. The mixture was kept in a freezer at −20° C. for 1 hourand then centrituged at 12,000×g, 4° C. for 20 minutes to obtain RNAprecipitate. The precipitate was suspended in 75% ethanol, centritugedin the same manner as above, and then dried for 10 minutes in a vaccum.The viral RNA precipitate was dissolved in 400 μl of TE buffer solution(10 mM Tris, pH 7.5, 1 mM EDTA) and used in the next step. The viral RNAsolution for later use may be kept at −70° C.

(1-B): Preparation of KHCV cDNA Library

(1-B-1): Preparation of KHCV cDNA

For preparation of cDNA, Zap-cDNA Synthesis Kit (Stratagene Inc., USA)was used. The hepatitic C viral RNA prepared in Example (1-A) was usedas a template for reverse transcriptase, and oligo-d(T) primer (SEQ IDNO: 2) having the nucleotide sequence of5′-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAG(T)₁₈-3′ and a random primer havingthe nucleotide sequence of5′-TTTTTCATGATTGGTGGTGGAACTGGACCGTCTCGAGNNNNNN-3′ wherein Ns may be thesame or different and each is A,T,C or G (hereinafter to be referred as“RANPSHCV” (SEQ ID NO: 1)) synthesized using a DNA synthesizer (AppliedBiosystems Inc., U.S.A., Model 380 B) were used.

A first strand of cDNA was prepared as follows. 18 μl of hepatitis viralRNA solution prepared in Example (1-A) was mixed with 2 μl of 0.1MCH₃HgOH, and the mixture was stood for 10 minutes at a room temperatureto unfold a secondary structure of RNA. 2 μl of 1M β-mercaptoethanol wasadded thereto and the mixture was kept for 5 minutes at roomtemperature. To the treated RNA solution were added 5 μl of reversetranscriptase reaction buffer solution (500 mM Tris-HCl, pH 8.3, 750 mMKCl, 30 mM MgCl₂, 10 mM dithiothreitol (DTT)), 2.5 μl of 10 mM each ofdATP, dGTP, dTTP and 5-methyl-dCTP, 2 μl of oligo-d(T) primer (1.4μg/μl) or 2 μl of RANPSHCV (1.0 μg/μl), 15 μl of distilled water treatedwith diethylpyrocarbonate (DEPC) and 1.0 μl of RNase inhibitor (1unit/μl, Promega Inc., USA), in this order; the mixture was stood for 10minutes at room temperature to the primers to the template; and then 2.5μl of MMLV reverse transcriptase (18 units/μl, Superscript RNase H⁻reverse transcriptase, BRL Inc., Cat. No. 8853SA) was added thereto. Thereaction mixture was incubated for 1 hour at 37° C. to synthesize thefirst strand of cDNA.

A second strand of cDNA was prepared as follows: To 45 μl of the firststrand solution so obtained were added 40 μl of 10×second strand buffersolution (188 mM Tris-HCl, pH 6.9, 906 mM KCl, 46 mM MgCl₂, 1.5 mM β-NAD(nicotinamide adenine dinucleotide), 100 mM (NH₄)₂SO₄), 6.0 μl of 10 mMdNTP's mixture (10 mM each of DATP, dCTP, dTTP and dGTP) and 298 μl ofdistilled water in order, and 1.0 μl of RNase H (4 units/μl) and 10.0 μlof DNA polymerase I (11 units/μl) were then dropped along the wall ofthe test tube. After instantly mixing it, the reaction mixture was thenincubated for 2.5 hours at 16° C.

The reaction solution was subjected to extraction with a same volume ofphenol-chloroform (1:1(v/v), phenol being already saturated with 0.5MTris-HCl (pH 7.5) and 0.1% (v/v) β-mercaptoethanol), 3 times. The upperaqueous phase was taken and mixed with 0.1 volume of 3M sodium acetateand 2-fold volume of 100% ethanol. The mixture was stood at −20° C.overnight and centrituged at 12,000×g, 4° C. for 20 minutes to obtainthe cDNA precipitate.

(1-B-2): Preparation of cDNA Library

In order to make the double stranded cDNA prepared in Example (1-B-1)into a blunt ended one, the cDNA precipitate was dissolved in 43.5 μl ofdistilled water. 39 μl of the cDNA solution was taken and then mixedwith 5.0 μl of T4 DNA polymerase reaction solution (670 mM Tris-HCl, pH8.8, 166 mM (NH₄)₂SO₄, 67 mM MgCl₂, 100 mM β-mercaptoethanol, 67 μMEDTA), 2.5 μl of 2.5 mM dNTP's mixture and 3.5 μl of T4 DNA polymerase(2.9 units/μl). The reaction mixture was stood for 30 minutes at 37° C.and the resulting product was extracted with phenol-chloroform andprecipitated with ethanol in the same manner as in Example (1-B-1).

In order to introduce a recognition site for restriction enzyme Eco RIat 5′-end, the blunt-ended double stranded cDNA prepared above wastreated as follows: To the blunt-ended cDNA were added 7.0 μl of Eco RIadaptor (Stratagene Inc., Zap-cDNA Synthesis Kit Cat. No. 200400,Calif., U.S.A.), 1.0 μl of 10×ligation buffer solution, 1.0 μl of T4 DNAligase (1000 units/μl) and 1.0 μl of 10 mM ATP, and was stood overnightat 4° C. The resulting mixture was then heated to 70° C. for 10 minutesto inactivate the ligase.

The cDNA so obtained may be directly subjected to a cloning. In thepresent example, however, said cDNA was amplified and then used in thecloning step.

For the amplification of cDNA, its PCR was carried out as follows: Tothe cDNA solution prepared above were added 10 μl of 10×PCR buffersolution (200 mM Tris-HCl, pH 8.3, 15 mM MgCl₂, 250 mM KCl, 0.5% Tween20, 1 mg/ml gelatin), 10 μl of 2 mM dNTP's mixture, 5 μl of primer,PSHCV (SEQ ID NO: 4) having the nucleotide sequence of5′-TTTTTCATGATTGGTGGTGGA-3′ and 5 μl of upper strand(5′-CCCCCCGAATTCGGCACGAG-3′) of the Eco RI adaptor (SEQ ID NO: 3), 1 μl(2.5 units) of Taq DNA polymerase (Perkin Elmer-Cetus Inc., 761 MainAvenue, Norwalk, Conn. 06859-0010, U.S.A.) and 69 μl of distilled water;and the PCR was then carried out using a thermal cycler (PerkinElmer-Cetus Inc., USA) which was programmed to repeat 25 times the cycleof: 95° C. for 30 seconds→55° C. for 30 seconds→72° C. for 2 minutes.After completing the reaction, the residual primers and dNTPs wereremoved using Centricon 100 (Amicon Inc., Cat. No. 4200, P.O. Box 91954,Chicago, Ill. 60693, U.S.A.). The product so obtained was extracted withphenol-chloroform and preciptated with ethanol in the same manner asabove, and then dissolved in 16 μl of TE buffer solution.

To the resulting solution were added 2 μl of 10×buffer solution (0.5MNaCl, 0.5M Tris-HCl, 50 mM MgCl₂, 5 mM DTT, pH 7.9) and 1 μl of each ofEco RI and Xho I (New England Biolabs Inc., 30 Tozer Rd., Berverly,Mass., U.S.A.), and the reaction mixture was then stood for 10 minutesat 37° C. to digest the cDNA partially. The cDNA fragments wereextracted with phenol-chloroform and precipitated with ethanol in thesame manner as above, and then dissolved in 10 μl of TE buffer solution.

The cDNA fragment so obtained was cloned into vector UNI-ZAPXR asfollows. To 10 μl of Eco RI-Xho I digested cDNA fragments solutionobtained above were added 2.0 μl of 10×ligation buffer solution, 2.0 μlof 10 mM ATP, 4 μl of of vector UNI-Z APXR solution (1 μg/μl) alreadytreated with Eco RI/Xho I and 2.0 μl of T4 DNA ligase (4 Weissunits/μl); and the reaction mixture was then incubated for 10 hours at16° C.

(1-B-3): In Vitro Packaging of the Vector Containing cDNA into Phase andAmplification of the cDNA Library

In order to package the ligated DNA prepared in Example (1-B-2) intophage, 10 μl of the final solution obtained in Example (1-B-2) was addedto Gigapack II Gold Packaging Extract (Stratagene Inc., U.S.A.) and thereaction mixture was stood for 2 hours at room temperature.

To the resulting mixture were added 500 μl of phage diluting solution(5.8 g of NaCl, 2.0 g of MgSO₄.7H₂O, 50 ml of 1M Tris-HCl, pH 7.5, 5 mlof 2% gelatin per liter) and 20 μl of chloroform (see Kretz et al.,Nucl. Acid. Res., 17, 5409(1989)).

The infection and amplification were carried out as follows. PLK-F′(Stratagene Inc., Zap-cDNA Synthesis Kit Cat. No. 200400), E. colimerA⁻, merB⁻ strain, was cultured in LB medium (10 g of Bacto-trypton, 5g of yeast extracts, 10 g of NaCl per liter) until O.D.₆₀₀ (OpticalDensity at 600 nm) reached 0.5. The cultured cells were precipitated anddissolved in 10 mM MgSO₄, adjusting their O.D.₆₀₀ to 1.0. 600 μl of thesolution was mixed with 200 μl of packaging mixture; the reactionmixture was then stood for 15 minutes at 37° C. to allow the phages toinfect into E. coli. To the resulting E. coli was added 6.5 ml of 0.7%NZY agar (7 g of NZ amines, 5 g of NaCl, 2 g of MgSO₄.7H₂O, 5 g of yeastextracts, 7 g of bactoagar per liter) melted and kept to 48° C.; and themixture was applied on a 150 mm-diameter NZY agar plate (7 g of NZamines, 5 g of NaCl, 2 g of MgSO₄7H₂O, 5 g of yeast extracts, 16 gbacto-agar per liter) and then incubated for 5 to 8 hours at 37° C. togenerate the phage plaques.

10 ml of phage diluting solution was poured onto the plate; the platewas shaken mildly for 15 hours at 4° C. to dissolve the phages; and theresulting mixture was centrifuged at 4,000×g to precipitate E. colicells, which were then removed off. To the HCV cDNA library solution soobtained was added 0.3% volume of chloroform; and the titer of the cDNAlibrary was determined to be about 10¹⁰ to 10¹³ PFU (plaque formingunits)/ml. 100% DMSO (dimethyl sulfoxide) was added thereto be make aconcentration of 7% (v/v); and the cDNA library was kept at −70° C.

(1-C): Screening of the cDNA Library by Immunoassay and Determination ofcDNA Sequence.

The cDNA library was screened by the immunoscreening method disclosed byHuynh, T. V. et al., DNA Cloning Techniques: A Practical Approach (D. M.Glover, ed.), pp 49-78, IRL Press, Oxford (1985), using the HCV antibodypurified from the supernatant after ultracentrifuging the 6-fold dilutedserum prepared in Example (1-A) by protein G affinity columnchomatography (Genex Inc., U.S.A.).

The cDNA library solution prepared in Example (1-B-3) was diluted to be50,000 PFU per 150 mm-diameter of plate; the diluted cDNA librarysolution was mixed with 600 μl of E. coli XL-1 blue (Stratagene Inc.,U.S.A., Zap-cDNA Synthesis Kit Cat. No. 200400) culture (O.D.₆₀₀=0.5)prepared by the same method as in Example (1-B-2) and 6.5 ml of 0.7% NYZagar was added thereto. Each mixture was applied on a 40 NZY agar plateand cultured for 12 hours at 37° C. to produce 2×10⁶ phage plaques.

Thereafter, plaque lift membrane of nylon filters (Bio-Rad Inc., Cat.No. 162-163, USA) of 137 mm diameter were impregnated with 10 mM IPTG(isopropyl-β-D-thiogalactopyranoside) solution and then blot-dried onWhatman 3MM filter. Each filter was placed above the agar in a plate;and incubated for 3.5 hours at 37° C. Each of the filters blotted withthe phage plaques was then washed with 15 ml of washing solution (10 mMTris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). To the filters was added15 ml of blocking solution (1% bovine serum albumin, 20 mM Tris-HCl, pH7.5, 150 mM NaCl); and incubated with gentle shaking for 1 hour at roomtemperature. Each of the filters was then washed 5 times by mild shakingwith 15 ml of TBST buffer solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl,0.05% (v/v) Tween-20) for 5 minutes at room temperature. The filterswere put in 15 ml of the solution prepared by diluting the purified HCVantibody (final protein concentration: 8.2 mg/ml) 1:200 with TBS buffersolution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 1% (w/v) FBS(fetal bovine serum) with mild shaking for 1 hour at room temperature;and then washed 5 times with mild shaking in TBST buffer solution for 5minutes at room temperature, respectively. Each of the filters was putin 15 ml of solution prepared by diluting biotinylated-goat anti-humanIgG and avidin conjugated-alkaline phosphatase (Pierce Inc., USA. Cat.Nos. 31770C, 21321C) 1:2000 with TBS buffer solution containing 1% (w/v)FBS, with mild shaking for 1 hour at room temperature; and then washed 5times with gentle shaking in 15 ml of TBST buffer solution for 5 minutesat room temperature. Each of the filters was then blot-dried on Whatman3MM filter.

For the coloring reaction, each of the filters was reacted in 15 ml ofcoloring solution (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂, 5mg nitro blue-tetrazolium, 2.5 mg 5-bromo-4-chloro-3-indolyl phosphate)in a dark room at room temperature for 30 minutes. The purple-colored,positive phage plaques were confirmed with eyes, which were expected toexpress the cDNA encoding a recombinant HCV antigen. Each of the filterswas washed with TBS buffer solution once; and coloring stopping solution(20 mM Tris-HCl, pH 2.9, 1mM EDTA) was added thereto to stop thecoloring process. Each of the filters was dried at room temperature andthen recorded on polaroid film.

Positive plaques were isolated; and incubated in 1 ml of phage dilutingsolution (10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂) for 1 to 2 hours at roomtemperature. The above immunoscreening assay was repeated to obtain theclones as a single phage plaque.

Each of the phage plaques confirmed as haboring the recombinant HCV genewas put into a sterilized microfuge tube containing 500 μl of 5M buffersolution (5.8 g of NaCl, 2.0 g of MgSO₄, 50 μl of 1M Tris-HCl, pH 7.5, 5ml of 2% gelatin per liter); 20 μl of chloroform was added thereto; andthen the contents of the tube was cultured with shaking for 1 to 2 hoursat room temperature. 200 μl (>1×10⁵ phage particles) of the solution soobtained, 1 μl of helper phage R408 (>1×10⁶ PFU/ml, Stratagene Inc.,U.S.A.) and 20 μl of E. coli XL-1 cell supension (O.D.₆₀₀=1.0) weremixed, and then the mixture was incubated at 37° C. for 15 minutes. Tothe resulting culture was added 5 ml of 2×YT medium (10 g of NaCl, 10 gof yeast extract, 16 g of Bacto-tryptone per liter), which was thencultured with shaking for 3 hours at 37° C. and then heated to 70° C.for 20 minutes. The resulting culure was diluted 1:100; and 200 μl ofthe diluted culture was mixed with 200 μl of E. coli XL1-Blue cell(O.D.₆₀₀=1.0). After incubating at 37° C. for 1 hour, 100 μl of theresulting culture was applied on LB plates containing ampicillin (50μg/ml); and incubated at 37° C. for 10 hours to obtain pBluescriptphagemid colonies haboring double stranded cDNA.

In order to prepare single stranded DNA, pBluescript colonies obtainedabove were incubated in a LB agar medium containing antibiotictetracycline (12.5 μg/ml) and screened to obtain the positive coloniesagain; and the positive single colonies as obtained were incubated intetracycline⁺ LB broth medium (2 to 3 ml) over-night. The culture wasthen incubated in 0.3 ml of super liquid medium (35 g Bacto-tryptone, 20g yeast extract, 5 g NaCl, adjusted to pH 7.5 with NaOH) and culturedwith shaking at 37° C. The culture was infected with helper phage R408and the culturing was carried out for 8 hours until O.D.₆₀₀ reached 0.3.

In effectuating the infection, the ratio of phage:cell largely dependson the type of cDNA harbored in pBluescript, and may be 20:1, 10:1, 1:1or 1:10. The single stranded DNAs were extracted from the supernatant ofthe culture obtained above.

The isolation and purification of double stranded phagemid and singlestranded phagemid was carried out by the method proposed by Sambrook, J.et al. in Molecular Cloning, 1, 2.73-2.81, Cold Spring Harbor, N.Y.(1989).

The length of the cDNA fragment contained in each clone was determinedby digesting the double stranded phagemid with the restrictionendonucleases Eco RI and Xho I; and 3 clones having cDNA fragments withdifferent length were obtained.

The nucleotide sequences of the 3 recombinant cDNAs were determinedusing the purified single stranded recombinant pBluescript phagemid orthe double stranded pBluescript phagemid as a template and using M13-20mer, primer T7, primer KS, primer SK or primer T3 (Stratagene Inc., USA)in accordance with Sanger's method (Proc. Natl. Acad. Sci. U.S.A., 74,5405(1977)), in which the resulting cDNA fragments were named KHCV 426,KHCV 652 and KHCV 403, respectively (see FIGS. 1 to 3).

(1-D): Screening of Recombinant Phages Harboring KHCV cDNA UsingOligonucleotide Probe and Determination of Nucleic Acid Sequence

(1-D-1): Isolation of cDNA Clones Overlapping with KHCV 652

In order to screen those recombinant phages harboring HCV cDNA which hadnot been screened by the above immunoscreening method, plaquehybridization was carried out by using the method described by Benton,W. D. et al., Science, 196, 180(1977); Connor, B. J. et al., Proc. Natl.Acad. Sci. U.S.A., 80, 278(1983); and Jacob, K. et al., Nature, 313,805(1985), using as probes oligonucleotides P652a (SEQ ID NO: 5)(5′-TTCATACCCGTTGAGTCTATGGAAACTACT-3′) and P652b (SEQ ID NO: 6)(5′-GCCATTCCAAGAAGAAGTGTGACGAACTCG-3′) whose nucleotide sequences wereselected from the nucleotide sequence of the cDNA KHCV 652 determined inExample (1-C).

The cDNA library solution prepared in Example (1-B) in an amountcontaining 50,000PFU was taken and then mixed with 600 μl of E. coliXL1-blue (diluted for O.D.₆₀₀ to be 0.5) prepared in Example (1-B-3) andmixed with 0.7% NZY agar. The mixture was poured onto a 150 mm NZY plateand incubated at 37° C. for 12 hours. From the total 30 plates, 1.5×10⁶phage plaques were obtained.

Thereafter, 137 mm-diameter-Nylon filters were carfully put on theplates, respectively, to blot the plaques to the filters. The nylonfilters were then removed and dried in air.

Each of the dried filters was placed on Whatman 3MM paper saturated with0.2M NaOH/1.5M NaCl for 1 to 2 minutes; and on Whatman 3MM papersaturated with 0.4M Tris-HCl, pH 7.6 and 2×SSC (SSC: 17.53 g of NaCl,8.82 g of sodium citrate, pH 7.0 per liter) for 1 to 2 minutes; and thendried in a vacuum oven at 80° C. for 2 hours.

After the drying, filters were washed with 500 ml of 3×SSC/0.1% SDSsolution at room temperature 3 to 4 times; and washed with the samesolution at 65° C. for 2 hours. Each of the filters was prehibridized in500 ml of prehybridization solution (6×SSC, 5×Denhardt solution (0.2 gof Ficoll, 0.2 g of polyvinylpyrrolidone, 0.2 g of BSA per liter), 0.05%sodium pyrophosphate, 100 μg/ml of boiled herring sperm DNA, 0.5% SDS)for 1 hour at 37° C. The filters were moved into hydridization solution(6×SSC, Denhardt solution, 100 μg/ml yeast tRNA, 0.05% sodiumpyrophosphate); and 30 ng of each of P652a and P652b labeled with ³²Pwas added thereto. The hybridization reaction was carried out for 24hours at 48° C.

The probes used above were labeled as follows. To a mixture of 32 ng ofprobe, 7.5 μl of 10×T4 kination buffer solution (0.5M Tris-HCl, pH 7.5,0.1M MgCl₂, 50 mM DTT, 0.5 mg/ml BSA), 100 μCi (γ-³²P)ATP and 50 unitsof T4 nucleotide kinase was added distilled water in a total volume of75 μl. The kination reaction was carried out for 30 minutes at 37° C.

After completing the hybridization, the filters were washed 5 times with6×SSC/0.05% sodium pyrophoshate solution for 10 minutes at roomtemperature; and once with the same solution for 30 minutes at 60° C.The washing was further carried out while raising the temperature by 2°C. over 15 minutes until the filter was confirmed to be completelywashed by checking with a Geiger counter (Ludlum Model 13). The washedfilters were exposed to X-ray film (Kadak X-Omat AR) for 24 to 48 hoursat −70° C.

The plaques confirmed as positive were screened off in the same manneras described above to obtain the plaques as a single phage plaque.

From the positive plaques so obtained, the double stranded phagemid andthe single stranded phagemid were prepared and the nucleotide sequencewas determined by the same method used in Example (1-C).

The cDNA clones overlapping with KHCV652 were named as KHCV 752 andKHCV675, respectively; and their length, position, nucleotide sequenceand the amino acid sequence encoded therein are shown in FIGS. 1 to 3.

(1-D-2): Isolation of cDNA Overlapping with KHCV 426

Oligonucleotides P426a (SEQ ID NO: 7)(5′-ACGAGACCTCCCGGGGCACTCGCAAGCACC-3′) and P426b (SEQ ID NO: 8)(5′-CGTAATTTGGGTAAGGTCATCGACACCCTC-3′), which were modeled on the basisof the nucleotide sequence of KHCV 426 cDNA obtained in Example (1-C),were synthesied. Using the oligonucleotides P426a and P426b as probes,plaque hydridization was carried out in the same manner as in Example(1-D-1). The cDNA clone overlapping with KHCV426 was detected by thesame method as described in Example (1-C); and designated as KHCV 240,whose length, position and nucleotide sequence and amino acid sequenceencoded therein are shown in FIGS. 1 to 3.

(1-D-3): Isolation of cDNA Overlapping with KHCV 240

Oligonucleotide P240b (SEQ ID NO: 10)(5′-GTCCGGGTGCTGGAGGACGGCGTGAACTA-3′), which was modeled on the basis ofthe nucleotide sequence of KHCV 240 determined in Example (1-D-2), wassynthesized. Using the oligonucleotide P240b as a probe, the cDNAlibrary prepared in Example (1-B) was screened in the same manner as inExample (1-D-1). The cDNA clone so obtained containing about 110nucleotides overlapping with KHCV 240 was designated as KHCV 513; andits nucleotide sequence was determined by Sanger's method. The length,position and nucleotide sequence of KHCV 513 and the amino acid sequenceencoded therein are shown in FIGS. 1 to 3.

(1-D-4): Isolation of cDNA Overlapping with KHCV 513

Oligonucleotide P513b (SEQ ID NO: 10)(5′-CGCATGGCCTGGGATATGATGATGAACTGG-3′), which was modeled on the basisof the nucleotide sequence of KHCV 513 determined in Example (1-D-3),was synthesized. Using the oligonucleotides P513b as a probe, the cDNAlibrary prepared in Example (1-B) was screened in the same manner as inExample (1-D-1). The 810 bp of cDNA clone which comprises about 130 bpof nucleotides overlapping with KHCV 513 was named as KHCV 810; and itsnucleotide sequence was determined by Sanger's method. The length,position and nucleotide sequence of KHCV 810 and the amino acid sequenceencoded therein are shown in FIGS. 1 to 3.

(1-D-5): Isolation of cDNA Overlapping with KHCV 810

Oligonucleotide P810b (SEQ ID NO: 11)(5′-AAATGAGACGGACGTGCTGCTCCTTAAC-3′), which was modeled on the basis ofthe nucleotide sequence of KHCV 810 determined in Example (1-D-4), wassynthesized. Using the oligonucleotides P810b as a probe, the libraryprepared in Example (1-B) was screened in the same manner as in Example(1-D-1). The cDNA clone so obtained which comprises about 65 bp ofnucleotides overlapping with KHCV 810 was named KHCV 798; and itsnucleotide sequence was determined by Sanger's method. The length,position and nucleotide sequence of KHCV 798 and the amino acid sequenceencoded in KHCV 798 are shown in FIGS. 1 to 3.

(1-D-6): Isolation of cDNA Overlapping with KHCV 403

Oligonucleotides P403A (SEQ ID NO: 12)(5′-GTGAAGAATTCGGGGGCCGGAACCTGGCAT-3′) and P403B (SEQ ID NO: 13)(5′-GCTGACCTCATTGAGGCCAACCTCTTGT-3′), which were modeled on the basis ofthe nucleotide sequence of KHCV 403 determined in Example (1-D-5), weresynthesized. Using the oligonucleotides P403A and P403B as probes, thelibrary prepared in Example (1-B) was screened in the same manner as inExample (1-D-1). The cDNA clone so obtained which comprises about 160 bpof nucleotides overlapping with KHCV 403 was named KHCV 932; and itsnucleotide sequence was determined by Sanger's method. The length,position and nucleotide sequence of KHCV 932 and the amino acid sequenceencoded in KHCV 932 are shown in FIGS. 1 to 3.

(1-D-7): Isolation of cDNA Overlapping with KHCV 932

Oligonucleotide P932b (SEQ ID NO: 14)(5′-CCGGGACGTGCTTAAGGAGATGAAGGCGAA-3′), which was modeled on the basisof the nucleotide sequence of KHCV 932 determined in Example (1-D-6),was synthesized. Using the oligonucleotide P932b as a probe, the cDNAlibrary prepared in Example (1-B) was screened in the same manner as inExample (1-D-1). The cDNA clone so obtained which comprises about 185 bpof nucleotides overlapping with KHCV 932 was named KHCV 496; and itsnucleotide sequence was determined by Sanger's method. The length,position and nucleotide sequence of KHCV 496 and the amino acid sequenceencoded in KHCV 496 are shown in FIGS. 1 to 3.

(1-D-8): Isolation of cDNA Overlapping with KHCV 496

Oligonucleotide P496b (SEQ ID NO: 15)(5′-CGTGTATGCGAGAAGATGGCCCTTTATGAC-3′), which was modeled on the basisof the nucleotide sequence of KHCV 496 determined in Example (1-D-7),was synthesized. Using the oligonucleotide P496b as a probe, the libraryprepared in Example (1-B) was screened in the same manner as in Example(1-D-1). The cDNA clone of 847 bp which comprises about 160 bp ofnucleotides overlapping with KHCV 496 was named KHCV 847; and itsnucleotide sequence was determined by Sanger's method. The length,position and nucleotide sequence of KHCV 847 and the amino acid sequenceencoded in KHCV 847 are shown in FIGS. 1 to 3.

(1-D-9): Isolation of cDNA Overlapping to KHCV 847

Oligonucleotide P847b (SEQ ID NO: 16)(5′-TGCGTGGGAGACAGCTAGACACACTCCAG-3′), which was modeled on the basis ofthe nucleotide sequence on 3′-end side of KHCV 847 determined in Example(1-D-8), was synthesized. Using the oligonucleotide P847b as a probe,the cDNA library prepared in Example (1-B) was screened in the samemanner as in Example (1-D-1). The cDNA clone of 494 bp so obtained whichcomprises about 94 bp of nucleotides overlapping with KHCV 847 was namedKHCV 494; and its nucleotide sequence was determined by Sanger's method.The length, position and nucleotide sequence of KHCV 494 and the aminoacid sequence encoded in KHCV 494 are shown in FIGS. 1 to 3.

(1-E): Preparation of cDNA by PCR

(1-E-1): Preparation of the KHCV cDNA Between KHCV 798 and KHCV 752

In order to clone the HCV cDNA between the 3′-end of KHCV 798 and the5′-end of KHCV 752, primers P798b (SEQ ID NO: 17)(5′-CTGGTTCCCGGAGCGGCATAC-3′) modeled on the basis of the nucleotidesequence on the 3′-end side of KHCV 798 and P752a (SEQ ID NO: 18)(5′-CCAGGTGATGACTTTGGTCTCCAT-3′) modeled on the basis of the nucleotidesequence on the 5′-end side of KHCV 752 were synthesized. Using theprimers P798b and P752a and the cDNA library prepared in of Example(1-B-1) using the primer of RANPSHCV, the polymerase chain reaction wascarried out as in Reference Example 7. After completing the reaction,some of the resulting mixture was subjected to 5% polyacrylamide gelelectrophoresis (PAGE) to confirm the amplification of the cDNA. To theremaining mixture was added 10 units of Klenow fragment, a DNApolymerase; and the reaction mixture was incubated for 30 minutes at 37°C. to make both ends to be blunt. The reaction mixture was subjected toPAGE and the DNA was electrically eluted to isolate the pure DNA. Thepurified DNA fragment was cloned into phage M13mp18 and its nucleotidesequence was determined. The DNA so obtained was named KHCV 570; and itsnucleotide sequence and the amino acid sequence encoded therein areshown in FIGS. 1 to 3.

KHCV 240 prepared in Example (1-D-2), KHCV 513 prepared in Example(1-D-3), KHCV 810 prepared in Example (1-D-4), KHCV 798 prepared inExample (1-D-5) and KHCV 570 prepared above overlapped in part eachother; and thus they were connected into a long open reading frame,which was named KHCV 2661.

(1-E-2): Preparation of KHCV cDNA Between KHCV 403 and KHCV 675

In order to clone a HCV cDNA fragment lying between KHCV 403 prepared inExample (1-C) and KHCV 675 prepared in Example (1-D-1), primers P675b(SEQ ID NO: 19) (5′-TCGATTCTTCGGTCCTGTGTGAGTGT-3′) and P675b₂ (SEQ IDNO: 20) (5′-AAAAAGAATTCGGATCCATGACGCGGGTTGTGCGTGGTAC-3′) modeled on thebasis of the nucleotide sequence on the 3′-end side of KHCV 675 andP403a₂ (SEQ ID NO: 21) (5′-CCCCCTCAGAGTCGACTCACTTCACGTTGTCAGTGGTCAT-3′)modeled on the basis of the nucleotide sequence on the 5′-end side ofKHCV 403 were synthesized. Using the primers P675b, P675b₂ and P403a₂prepared above and P403a prepared in Example (1-D-6), PCR was carriedout as follows.

To a mixture of 0.2 μg of P674b, 0.2 μg of P403a, 2 μl of cDNA preparedin Example (1-B-1) using the random primer RANPSHCV, 10 μl of 10×PCRbuffer solution, 10 μl of 2 mM dNTP's mixture, and 2.5 units of Taqpolymerase was added distilled water to adjust the total volume to be100 μl. The mixture was subjected to a first PCR by repeating 10 timesthe cycle of: 95° C. for 2 minutes→55° C. for 2 minutes→72° C. for 3minutes. After adding 2 μg of P675b₂ and 2 μg of P403a₂ to the resultingmixture, the second PCR was carried out by repeating 20 times the abovethermal cycle.

After completing the reaction, the amplification of the cDNA wasconfirmed; and the sequence of the cDNA was determined in the samemanner as in Example (1-E-1). The cDNA so obtained was named KHCV 1774,and its nucleotide sequence and the amino acid sequence encoded thereinare shown in FIGS. 1 to 3.

(1-E-3): Cloning of 3′-end Region of KHCV cDNA and Determination ofNucleotide Sequence Thereof

In order to clone cDNA corresponding to the 3′-end region of HCV genome,PCR using the primers RANPSHCV and DA17PSHCV (SEQ ID NO: 22)(5′-TGGTGGTGGAACTGGACCGTA₁,-3′) was carried out as follows.

Primer PSHCVSL (SEQ ID NO: 23), 5′-AAAAGTCGACTGGTGGTGGAACTGGACCGT-3′,contains 21 fixed nucleotides of primer RANPSHCV or DA17PSHCV of Example(1-B-1) and Sal I recognition site (5′-GTCGAC-3′); while primer KHCVR60(SEQ ID NO: 24), 5′-GTGTCCGCGCTAAGCTACTGTCC-3′, contains thosenucleotides designed from the nucleotide sequence of the 3′-end regionof KHCV 494 of Example (1-D-9). Using primers PSHCV and KHCVR60, a firstPCR was carried out in the same manner as in Reference Example 7.

In a second PCR, primer KHCVR61 (SEQ ID NO: 25)(5′-TGTGGCAAGTACCTCTTCAACTGG-3′) was synthesized. KHCVR61 consists of asequence complementary to the nucleotide sequence of the 3′-end regionof KHCV 494, and closer to the 3′-end than KHCVR60.

10 μl of KHCVR61 was added to the mixture resulted from the first PCR,and the second PCR was then carried out by the same method as inReference Example 7.

After completing the reaction, amplification of cDNA was confirmed andits nucleotide sequence was determined in the same manner as in Example(1-E-1). The cDNA so obtained, having 266 nucleotides, was named KHCV266. The position and nucleotide sequence of KHCV 266 and the amino acidsequence encoded therein are shown in FIGS. 1 to 3. In the nucleotidesequence of KHCV 266, two terminator codons were found, althoughpoly(A)⁺ tail was not found.

(1-E-4): Cloning of 5′-end Region of KHCV cDNA and Determination ofNucleotide Sequence

Using primer KHCVL69 (SEQ ID NO: 26) (5′-GTCCTGTGGGCGGCGGTTGGTGTTACG-3′)modeled on the basis of the 5′-end side nucleotides of KHCV 426 preparedin Example (1-C), a single stranded cDNA was prepared in the same manneras in Example (1-B-1). 50 μl of the mixture resulted from the above wasdiluted with 1 ml of TE buffer solution (10 mM Tris-HCl, pH 7.5, 1 mmEDTA). The diluted mixture was concentrated to 10 μl by using Centricon100 (Amicon Inc., U.S.A., #4200) so as to remove the residual primersand dNTPs.

In order to make a poly d(T) tailed cDNA or poly d(G) tailed cDNA, to 10μl of the cDNA solution so obtained were added 4 μl of 5×tailing buffersolution (0.5M potassium cacodylate, pH 7.2, 10 mM CoCl₂, 1 mM DTT), 4μl of 1 mM dTTP (or 4 μl of 1 mM dGTP) and 10 units of terminaldeoxynucleotide transferase (BRL Inc., U.S.A., #80085B); and distilledwater was added to adjust the total volume to be 50 μl. The reactionmixture was stood for 30 munutes at 37° C. and then heated to 65° C. for5 minutes.

The poly d(T)⁺ tailed cDNA (or the poly d(G) tailed cDNA) so obtainedwas amplified by PCR using primers KHCVL70 (SEQ ID NO: 27)(5′-TTGAGGTTTAGGATTCGTGCTCAT-3′) (or dC12R1R0 (SEQ ID NO: 28);5′AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGA(C)₁₂-3′), dT17R1R0 (SEQ IDNO: 29) (5′-AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGA(T)₁₇-3′), R0 (SEQID NO: 30) (5′-AAGGATCCGTCGACATC-3′) and R1 (SEQ ID NO: 31)(5′-GACATCGATAATACGACTCAC-3′) designed from the nucleotide sequence ofKHCV 426 prepared in Example (1-C).

To 2 μl of cDNA solution were added 5 μl of 10×Taq polymerase buffersolution (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl₂, 0.1%gelatin), 5 μl of 1.5 mM dNTPs mixture, 2.0 μg of KHCVL69 and 2.0 μg ofdT17R1R0; and distilled water was added to adjust the total volume to be50 μl. The mixture was heated to 95° C. for 7 minutes and then cooled to75° C. 2.5 units of Taq DNA polymerase was added thereto; and 30 μl ofmineral oil was then added to prevent evaporation thereof. The reactionmixture was cooled to 45° C. for 2 minutes to allow the primers tocomplementarily bind the single stranded cDNA, and then reacted at 72°C. for 22 minutes. A first PCR was carried out by repeating 30 times thecycle of: 95° C. for 45 seconds→4 50° C. for 25 seconds→72° C. for 2minutes; and finally at 72° C. for 15 minutes.

2 μg of primer R0 (or R1) and 2 μg of KHCVL70 were added to 10 μl of themixture resulted from the above; and a second PCR was carried out byrepeating 30 times the same cycle as above. After completing thereaction, amplification of the cDNA, having 380 bp, was confirmed andits nucleotide sequence was determined in the same manner as in Example(1-E-1). The cDNA clone so obtained was named KHCV 366, and the positionand nucleotide sequence of KHCV 366 and the amino acid sequence encodedtherein are shown in FIGS. 1 to 3.

The KHCV cDNA clones obtained in Example 1 connected to a full lengthKHCV cDNA having 9372 nucleotides; and the full length cDNA was namedKHCV-LBC1, which was deposited with ATCC on May 14, 1991 with theaccession No. of 75008.

EXAMPLE 2 Preparation of HCV Subtype cDNA

(2-A): Extraction of RNA

To 100 μl of each serum collected from 13 Korean patients with hepatitisC (Samples #2, #3, #20, #21, #23, #25, #26, #27, #28, #29, #30, #31 and#32) was added 300 μl of RNAzol B (Cinna/Biotecx, P.O. Box 1421,Friendwood, Tex., U.S.A.) to disrupt the cells; and the KHCV RNAs werethen extracted in the same manner as in Example (1-A). The KHCV RNAsextracted from these 13 samples were named LBC2, LBC3, LBC20, LBC21,LBC23, LBC25, LBC26, LBC27, LBC28, LBC29, LBC30, LBC31 and LBC32,respectively.

(2-B): Preparation of cDNA

Using the HCV RNAs prepared in Example (2-A) as templates and randomprimers (5′-NNNNNN-3′, wherein Ns may be the same or different and maybe G, A, T or C with the same proportion) as primers for reversetranscriptase, the HCV cDNAs were prepared in the same manner as inExample (1-B-1). The cDNAs so obtained were named KHCV-LBC2 cDNA,KHCV-LBC3 cDNA, KHCV-LBC20 cDNA, KHCV-LBC21 cDNA, KHCV-LBC23 cDNA,KHCV-LBC25 cDNA, KHCV-LBC26 cDNA, KHCV-LBC27 cDNA, KHCV-LBC28 cDNA,KHCV-LBC29 cDNA, KHCV-LBC30 cDNA, KHCV-LBC31 cDNA and KHCV-LBC32 cDNA,respectively.

(2-C): Amplification of KHCV cDNA by PCR

(2-C-1): Design of Primers

The primers for the amplification of the NS2 and the NS5 regions of HCVcDNAs were designed from the regions relatively commonly present in thenucleotide sequences of the Japanese type reported by Kato et al., Proc.Natl. Acad. Sci. USA, 87, 9524-9528 (1990) and Takamizawa et al., J.Virol., 65, 1105-1113(1991); of the American type reported by Choo etal., Sicence, 244, 359-363 (1989); and of the KHCV-LBC1 prepared inExample 1. The positions of the nucleotide sequences prepared above werenumbered on the basis of the nucleotdies sequence of KHCV-LBC1.

Primers for Amplification of NS2 Region of HCV cDNA

NS2S1 (SEQ ID NO: 32) (5′-CGGGAGATGGCCGCATCGTG-3′) corresponded to thestrand of the fragment from the 2776th to the 2795th nucleotides inKHCV-LBC1; and NS2N1 (SEQ ID NO: 33) (5′-ACCTGCTAGTGCGGCCAGCTTCAT-3′)corresponded to the complementary strand of the fragment from the 3180thto the 3157th nucleotides of KHCV-LBC1, which were used in carrying outa first PCR for the amplification of the NS2 region of HCV cDNA. NS2S2(SEQ ID NO: 34) (5′-TTTTGGATCCGCGGTTTTTGTAGGTCTGGT-3′) corresponded tothe strand of the fragment from the 2803rd to the 2822 nd nucleotides inKHCV-LBC1, which had a BamH I recognition site for the convenience ofcloning; and NS2N2 (SEQ ID NO: 35) (5′-AAAGTCGACATGAAGACCATTTGGAC-3′)corresponded to the complementary strand of the fragment from the 3159thto the 3142 nd nucleotides in KHCV-LBC1, which had a Sal I recongnitionsite at its 5′-end for the convenience of cloning. NS2S2 and NSS2N2 wereused in carrying out a second PCR.

Primers for Amplifiction of NS5 Region of HCV cDNA

NS5S1 (SEQ ID NO: 36) (5′-ATGGGGATCCATATGACACCCGCTG(T/C)TTTGA-3′,wherein T/C means Thymines and Cytosines mixed in the ratio of 1:1), thenucleotide sequence from the 10th nucleotide (as counted from the 5′-endof NS5S1) to the 3′-end corresponded to the nucleotide sequence of thefragment from the 8252 nd to the 8273rd nucleotides in KHCV-LBC1. InNS5N1 (SEQ ID NO: 37) (5′-CCCCGTCGACCTAGTCATAGCCTCCGTGAA-3′), thenucleotide sequence from the 9th nucleotide to the 3′-end correspondedto the complementary strand of the fragment from the 8635th to the8614th nucleotides in KHCV-LBC1. Primer NS5N1 was used in carrying out afirst PCR for the amplification of the NS5 region.

In NS5S2 (SEQ ID NO: 38) (5′-TTTGAGGATCCACGGTCACTGAGAA(T/C)GACAT-3′,wherein T/C has the same meaning as above), the nucleotide sequence fromthe 12th nucleotide to the 3′-end corresponded to the strand of thefragment from the 8278th to the 8297th nucleotides in KHCV-LBC1, andNS5S2 had a BamH I recognision site at its 5′-end. Primer NS5S2 was usedin corrying out a second PCR.

The above primers were synthesized using DNA synthesizer (AppliedBiosystems Inc., Model 380 B, USA) employing automized solid phasephosphoamidite chemistry. The synthesized primers were isolated byelectrophoresis using denaturation polyacrylamide gel (2M urea, 12%acrylamide and bis acryamide (29:1, w/w) in 50 mM Tris, 50 mM boricacid, 1 mM EDTA-Na₂), and purified through C18 column chromatography(SEPAK; Waters Inc., USA) using a mixture of acetonitrile-water (50:50,v/v) as an eluent. The concentration of each primer was determined by anO.D. value at 260 nm.

(2-C-2): PCR for Amplification of NS2 Region of KHCV cDNA

A first PCR was carried out as follows. To 5 μl of each of KHCV-LBC2cDNA, KHCV-LBC3 cDNA, KHCV-LBC20 cDNA, KHCV-LBC21 cDNA, KHCV-LBC23 cDNA,KHCV-LBC25 cDNA, KHCV-LBC26 cDNA, KHCV-LBC27 cDNA, KHCV-LBC28 cDNA,KHCV-LBC29 cDNA, KHCV-LBC30 cDNA, KHCV-LBC31 cDNA and KHCV-LBC32 cDNAprepared in Example (2-B) were added 10 μl of 10×Taq polymerase buffersolution (10 mM Tris-HCl, pH 8.3, 500 mM HCl, 15 mM MgCl₂, 0.1% (w/v)gelatin), 10 μl of 2 mM dNTP's mixture, 0.2 μg of NS2S1, 0.2 μg of NS2N1and 0.5 μl of AmpliTaq DNA polymerase (Perkin Elmer-Cetus, USA); anddistilled water was added to adjust the total volume to be 100 μl. Toeach of such solution, 50 μl of mineral oil was added to preventevaporation thereof. The first PCR was carried out by repeating 40 timesthe thermal cycle of: 95° C. for 2 minutes→55° C. for 2 minutes→72° C.for 3 minutes. The second PCR was carried out using 1 ml of first PCRproducts and 2 μg of NS2S2/NS2N2 primer set by repeating 25 times.

Each of the resulting mixtures was mixed with a same volume ofphenol/chloroform and then centrifuged to removed the residual enzymes.To each of the supernatants were added 0.1 volume of 3M sodium acetateand a 2.5-fold volume of absolute ethanol; and the resulting mixture wasthen centrifuged to yield 340 bp of double stranded DNA.

The DNA fragments from the 13 different templates were named NS2-LBC2,NS2-LBC3, NS2-LBC20, NS2-LBC 21, NS2-LBC 23, NS2-LBC25, NS2-LBC26,NS2-LBC27, NS2-LBC28, NS2-LBC29, NS2-LBC30, NS2-LBC31 and NS2-LBC32,respectively.

(2-C-3): PCR for Amplification of NS5 Region of HCV cDNA

Primers NS5S1 and NS5N1 were used to carry out a first PCR and PrimersNS5S2 and NS5N1 were used to carry out a second PCR in the same manneras in Example (2-C-2) to obtain 320 bp of DNA segments.

The resultant DNA fragments amplified from KHCV-LBC20 cDNA, KHCV-LBC21cDNA, KHCV-LBC23 cDNA, KHCV-LBC25 cDNA, KHCV-LBC26 cDNA, KHCV-LBC27cDNA, KHCV-LBC28 cDNA, KHCV-LBC29 cDNA, KHCV-LBC30 cDNA, KHCV-LBC31 cDNAand KHCV-LBC32 cDNA were named NS5-LBC20, NS5-LBC 21, NS5-LBC 23,NS5-LBC25, NS5-LBC27, NS5-LBC28, NS5-LBC29, NS5-LBC30, NS5-LBC31 andNS5-LBC32, respectively.

Each of the fragments was digested with Sal I and BamH I; the digestedfragment was cloned into M13mp19; and its nucleotide sequence wasdetermined by using Sanger's method. Each of the nucleotide sequences isshown in FIGS. 7 to 26, respectively.

EXAMPLE 3 Preparation of Vector for the Expression of KHCV cDNAFragments in Yeast

(3-A): Amplification of KHCV cDNA Fragments

(3-A-1): Preparation of Fragments K384, K510, K573, K897, K403 and K590

<Step 1>

In order to connect a ubiquitin gene to each of the KHCV cDNA fragmentscloned in Examples (1-C), (1-D) and (1-E) (hereinafter, the gene made byconnecting the ubiquitin gene to the KHCV cDNA fragments will bereferred to as “UB-KHCV”) and to clone the UB-KHCV into an expressionvector for yeasts, the primers disclosed below were synthesized.

Primer PCOREUBI (SEQ ID NO: 39)(5′-CTTGGTGTTGAGACTCCGCGGTGGTATGAGCACGAATCCTAAACC-3′) contains 25nucleotides on the 5′-end region overlapping with the 3′-end region ofthe ubiquitin gene; and the other nucleotides correspond to the regionfrom the 343rd to the 360th nucleotides of KHCV-LBC1.

Primer PSALCORE14 (SEQ ID NO: 40)(5′-GGGGTCGACTATTAGCATGTGAGGGTGTGGATGAC-3′) contains a stop codon tostop translation just after the 726th nucleotide and a recognition siteof Sal I.

Primer PSALCORE17 (SEQ ID NO: 41)(5′-GGGGTCGACTATTAGGGCAGATTCCCTGTTGCATA-3′) contains a stop codon tostop translation just after the 852 nd nucleotide and a recognition siteof Sal I.

Primer PSALCORE22 (SEQ ID NO: 42)(5′-GGGGTCGACTATTAAGCGGAACTGGGGATGGTCAA-3′) contains a stop codon tostop translation just after the 915th nucleotide and a recognition siteof Sal I.

Primer PK403UBI (SEQ ID NO: 43)(5′-CTTGGTGTTGAGACTCCGGTGGTACGGGCATGACCACTGACAA-3′) contains 25nucleotides on the 5′-end region which are the same as those ofPCOREBUI; and the other nucleotides are designed to initiate translationfrom the 6649th nucleotide of KHCV-LBC1.

Primer PK573UBI (SEQ ID NO: 44)(5′-CTTGGTGTTGAGACTCCGCGGTGGTACATGGACAGGCGCCCTGA-3′) contains 25nucleotides on the 5′-end region which are the same as those ofPCOREUBI; and the other nucleotides are designed to initiate translationfrom the 7612th nucleotide of KHCV-LBC1.

Primer PK403SAL (SEQ ID NO: 45)(5′-GACTGGTCGACTATTACTCTTGCCGCCACAAGAGGTT-3′) is designed to stoptranslation just after the 7050th nucleotide of KHCV-LBC1; and has arecognition site of Sal I and two stop codons (TAATAG).

Primer PK897UBI (SEQ ID NO: 46)(5′-CTTGGTGTTGAGACTCCGCGGTGGTGCGGTGGAATTCATACCCG-3′) contains 25nucleotides on the 5′-end region which are the same as those of PCOREUBIand the other nucleotides are designed to initiate translation from the3916th nucleotide of KHCV-LBC1.

Primer PK897SAL (SEQ ID NO: 47)(5′-GACTGGTCGACTATTAACACGTATTACAGTCGATCAC-3′) is designed to stoptranslation just after the 4713th nucleotide of KHCV-LBC1; and has arecognition site of Sal I and two stop codons (TAATAG).

Primer PK573SAL (SEQ ID NO: 48)(5′-GACTGGTCGACTATTAGTACTGGAATCCGTATGAGGAG-3′) is designed to stoptranslation just after the 8184th nucleotide of KHCV-LBC1; and has arecognition site of Sal I and two stop codons (TAATAG) on the 3′-endsite.

Primer P426B (SEQ ID NO: 49) (5′-GGGTGGGCAGGATGGCTCCTG-3′) consists ofthe region from the 616th to the 636th nucleotides of KHCV-LBC1.

Primer P240B (SEQ ID NO: 50) (5′-CCTGTTGCATAGTTCACGCCGT-3′) consists ofthe region from the 842 nd to the 821st nucleotides of KHCV-LBC1.

Primer P652B (SEQ ID NO: 51)(5′-GTCATTCCAAGAAGAAATGTGACGAGCTCGCTGCAAAG-3′) consists of the regionfrom the 4523rd to the 4560th nucleotides of KHCV-LBC1.

Primer P403B (SEQ ID NO: 52) (5′-GCTGACCTCATTGAGGCCAACCTCTTGT-3′)consists of the region from the 7012th to the 7039th nucleotides ofKHCV-LBC1.

<Step 2>

A single cDNA fragment was prepared from 3 clones, i.e., KHCV426,KHCV240 and KHCV 513 overlapping with each other, as follows. To amixture of 2.0 μg of PCOREUBI, 0.02 μg of P426B, 2 μg of P240B and 50 ngof KHCV-LBC1 DNA were added 10 μl of 1OX Taq polymerase buffer solution,10 μl of 10 nM dNTP's mixture and 2.5 units of Taq polymerase; anddistilled water was added thereto to adjust the total volume to be 100μl. A first PCR was then carried out by repeating 25 times the thermalcycle as in Reference Example 7. The resulting mixture was subjected to5% polyacrylamide gel electrophoresis to isolate 500 bp of the PCRproduct (hereinafter, referred as “PCR product A”). Thereafter, using 50ng of PCR product A and 50 ng of KHCV-LBC1 DNA as templates, and 2 μg ofPCOREUBI and 2 μg of PSALCORE22 as primers, a second PCR was carried outunder the same condition as in the above first PCR. The resultingmixture was subjected to 5% polyacrylamide gel electorphoresis toisolate 580 bp of the final product (hereinafter, referred to as “PCRproduct B”), which was then dissolved in 50 μl of TE buffer solution.

<Step 3>

In order to carry out further PCRs using the PCR product B obtained inStep 2 as a template, 3 different test tubes, i.e., Tube A containing 2μg of PCOREUBI and 2 μg of PSALCORE14, Tube B containing 2 μg ofPCOREUBI and 2 μg of PSALCORE17, and Tube C containing 2 μg of PCOREUBIand 2 μg of PSALCORE22, in addition to 50 ng of the PCR product B addedto each of the tubes, were prepared.

On the other hand, for PCRs using KHCV-LBC1 DNA as a template, other 3different test tubes, i.e., Tube D containing 2 μg of PK897SAL, 0.02 μgof P652B and 2 μg of PK897UBI, Tube E containing 2 μg of PK403SAL and 2μg of PK403UBI; and Tube F containing 2 μg of PK573SAL and 0.022 μg ofP403Bb and 2 μg of PK573UBI, in addition to 50 ng of KHCV-LBC1 added toeach of the tubes, were also prepared.

Thereafer, to each of Tubes A to F were added 10 μl of 10×Taq polymerasebuffer solution, 10 μl of 10 mM dNTP's mixture and 25 units of Taqpolymerase; and distilled water was added thereto to adjust the totalvolume to be 100 μl. The PCRs were carried out under the same conditionas in Step 2.

<Step 4>

The PCR products obtained in Step 3 were subjected to 5% polyacrylamidegel electrophoresis. As a result, it was confirmed that 384 bp DNAfragment was produced in Tube A, 510 bp of DNA in Tube B, 573 bp of DNAin Tube C, 798 bp of DNA in Tube D, 402 bp of DNA in Tube E, and 573 bpof DNA in Tube F were amplified. The DNA fragments were purified by thesame polyacrylamide gel electrophoresis as above; and named fragmentK384, fragment K510, fragment K573, fragment K897, fragment K403 andfragment K590, respectively.

(3-A-2): Preparation of cDNA Fragment Encoding KHCV Envelope Protein

<Step 1>

In order to connect the synthesized ubiquitin gene to each of E 2N geneand E 2C gene, which corresponds to the region from the 1510th to the2010th nucleotides and the region from the 2011th to the 2529thnucleotides of KHCV-LBC1, respectively, and to clone each into anexpression vector of yeasts, the following primers were synthesized.

Primer PE2NUBI (SEQ ID NO: 52)(5′-CTTGGTGTTGAGACTCCGCGGTGGTGGGGCGCAAGGTCGGGCCGCT-3′) contains 25nucleotides on the 5′-end region overlapping with the 3′-end region ofubiquitin gene; and the other nucleotides correspond to the region fromthe 1510th to the 1530th nucleotides of KHCV-LBC1.

Primer PE2NSAL (SEQ ID NO: 53)(5′-GACTGGACTATTAATTCATCCAGGTAGAACCGAACCA-3′) contains a stop codon tostop translation just after the 2010th nucleotide of KHCV-LBC1; and arecognition site of Sal I.

Primer PE2CUBI (SEQ ID NO: 54)(5′-CTTGGTGTTGAGACTCCGCGGTGGTGGCACTGGGTTCACCAAGACA-3′) contains 25nucleotides on the 5′-region overlapping with the 25 nucleotides on the3′-end region of ubiquitin gene; and the other nucleotides correspond tothe region from the 2011th to the 2031th nucleotides of KHCV-LBC1.

Primer PE2CSAL (SEQ ID NO: 55)(5′-GACTGGACTATTACGCGTCCGCCAGAAGAAGGAAGAG-3′) contains a stop codon tostop translation after the 2529th nucleotide of KHCV-LBC1; and arecognition site of Sal I.

<Step 2>

Tube A was provided with 2 μg of each of PE2NUBI and PE2NSAL, and Tube Bwas provided with 2 μg of each of PE2CUBI and PE2CSAL. To each of TubesA and B were added 50 μg of KHCV-LBC1, 10 μl of 10×polymerase buffersolution, 10 μl of 10 mM dNTP's mixture and 2.5 units of Taq polymerase;and distilled water was added thereto to adjust the total volume to be100 ml. The PCRs were carried out by repeating 25 times the same thermalcycle as in Reference Example 7.

<Step 3>

The PCR products obtained in Step 2 were subjected to 5% polyacrylamidegel electrophoresis. As a result, it was confirmed that 501 bp of DNA inTube A and 519 bp of DNA in Tube B were amplified, respectively. TheDNAs were purified by the same polyacrylamide gel electrophoresis asabove and named segment E2N and segment E2C, respectively.

(3-B): Preparation of Expression Vector for Yeast

(3-B-1): Preparation of pYLBC-A/G-UB-CORE14, PYLBC-A/G-UB-CORE17,pYLBC-A/G-UB-CORE22, pYLBC-A/G-UB-KHCV897, PYLBC-A/G-UB-KHCV403 andPYLBC-A/G-UB-KHCV573

2 μg of plasmid pYLBC-A/G-UB-HGH(ATCC74071) was completely digested withPst I and Sal I in NEB buffer solution 3, while 2 μg of the same plasmidwas completely digested with Pst I and Sac II in NEB buffer solution 4referred to in Reference Example 1. The resulting mixtures weresubjected to 0.7% agarose gel electrophoresis to isolate 9.8 kb fragmentand 3.4 kb fragment, which were named fragments PL2 and PT2,respectively.

Among the fragments of K384, K510, K573, K987, K403 and K590 prepared inExample (3-A-1), fragments K897, K403 and K590 were completely digestedwith Sal I and Sac II in NEB buffer solution 3; fragments K384, K510 andK573 were completely digested with Sal I in NEB buffer solution 3,respectively. The products were extracted with phenol/chloroform andprecipitated with ethanol; and dissolved in 20 μl of TE buffer solution.Fragments K384, K510 and K573 were further partially digested with SacII in NBE buffer solution 4 for 10 minutes; and the products wereextracted with phenol/chloroform and precipitated with ethanol; anddissolved in 20 μl of TE buffer solution.

The above fragments were used in the ligation as follows. Ligation TubeA was provided with 100 ng of fragment K384; Ligation Tube B wasprovided with 100 ng of fragment K510; Ligation Tube C was provided with100 ng of fragment K573; Ligation Tube D was provided with 100 ng offragment K897; Ligation Tube E was provided with 100 ng of fragmentK403; and Ligation Tube F was provided with 100 ng of fragment K573. Toeach of the tubes were added 100 ng of fragment PL2, 100 ng of fragmentPT2, 2 μl of 10×ligation buffer solution and 10 units of T4 DNA ligase;and distilled water was added to adjust the total volume to be 20 μl.The ligation was carried out for 12 hours at 16° C.

E. coli HB101(ATCC 33694) was transformed with each of the ligatedvectors respectively.

The vector containing K384 was isolated and named PYLBC-A/G-UB-CORE14;the vector containing K510 was isolated and named pYLBC-A/G-UB-CORE17;the vector containing K573 was isolated and named pYLBC-A/G-UB-CORE22;the vector containing K897 was isolated and named pYLBC-A/G-UB-KHCV897;the vector containing K403 was isolated and named pYLBC-A/G-UB-KHCV403;and the vector containing K590 was isolated and namedpYLBC-A/G-UB-KHCV573 (see FIGS. 30).

(3-B-2): Preparation of pYLBC-A/G-UB-E2N and pYLBC-A/G-UB-E2C

2 μg of plasmid pYLBC-A/G-UB-HGH(ATCC 74071) was completely digestedwith Pst I and Sal I in NEB buffer solution 3, and 2 μg of the sameplasmid was completely digested with Pst I and Sac II in NEB buffersolution 4. The resulting mixtures were subjected to 0.7% agarose gelelectrophoresis to isolate 9.8 kb and 3.4 kb fragments, which were namedfragment PL2 and fragment PT2, respectively.

Each of fragments E2N and E2C prepared in Example (3-A-2) was completelydigested with Sac II in NEB buffer solution 4 and further partiallydigested with Sal I in NEB buffer solution 3. Each of the products wasextracted with phenol/chloroform and precipitated with ethanol; anddissolved in 20 μl of TE buffer solution. The fragments were namedfragment E2N-T2/L and fragment E2C-T2/L, respectively.

Ligation Tube G was provided with 100 ng of E2N-T2/L and Ligation Tube Fwas provided with 100 ng of E2C-T2/L. To each of the tubes were added 10ng of PL2, 10 ng of PT2, 2 μl of 10×ligation buffer solution and 10units of T4 DNA ligase; and distilled water was added to adjust thetotal volume to be 20 μl. The reaction was carried out for 12 hours at16° C. E. coli HB101(ATCC 33694) was transformed with each of theligated vectors. The vector containing fragment E2N-T2/L was namedpYLBC-A/G-UB-E2N; and the vector containing fragment E2C-T2/L was namedpYLBC-A/G-UB-E2C (see FIG. 30).

(3-C): Transformation of Yeast and Production of Protein

Yeasts were transformed with the expression vectors prepared in Example(3-B-2) by the same method as in Reference Example 5. Of the transformedyeasts, Saccharomyces cerevisiae DC 04 transformed withpYLBC-A/G-UB-KHCV403 (S. cerevisiae pYLBC-A/G-UB-KHCV 403) was depositedwith accession number of ATCC 74079 on Jun. 27, 1991; and Saccharomycescerevisiae DC 04 transformed with pYLBC-A/G-UB-CORE14 (S. cerevisiae DC04-UB-CORE 14) was deposited with the accession number of ATCC 74081 onJul. 1, 1991; and Saccharomyces cerevisiae DC 04 transformed withpYLBC-A/G-UB-E2C (S. cerevisiae DC 04-UB-E2C) was deposited with theaccession number of ATCC 74117 on Dec. 11, 1991, to American TypeCulture Collection under the terms of Butapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure.

Among the transformed yeasts, Saccharomyces cerevisiae DC 04-UB-KHCV403was cultured in 3 ml of leucine-deficient medium (6.7 g of yeastnitrogen base without amino acids (Difco Inc., U.S.A.), 2.5 g of aminoacids mixture without leucine per liter, and 5% glucose) at 30° C.overnight. The culture was transferred into 100 ml of YEPD medium (2%peptone, 1% yeast extracts, 2% glucose) and cultured at 30° C. overnightto produce the KHCV protein. The resulting culture had the O.D. value,at 650 nm, of about 25. The other transformed yeasts were cultured inthe same manner as above to produce the KHCV proteins.

Each of the cultures was harvested the amount corresponding to theO.D.₆₅₀ value of 10; and centrifuged. Each of the precipitates wassuspended in 400 μl of buffer solution (10 mM Tris-HCl, pH 7.5, 1 mMEDTA, 2 mM PMSF (phenylmethylsulfonyl fluoride), 8M urea); and thenvigorously shaken with a same volume of glass beads (diameter 0.4 mm) todestroy the cell walls. The yeast extracts so obtained were subjected to15% sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(SDS-PAGE) by employing Laemmli's method (Laemmli et al., Nature, 277,680(1970)); and the gel was stained with Coomassie brilliant blue R250to confirm the production of KHCV proteins (see FIG. 31-A).

The proteins separated on the gel were blotted onto a nitro cellulosefilter. The filter was put in PBS (10 mM phosphate, pH 7.0, 0.15M NaCl)containing 0.2% Tween 20; and shaken for 2 hours at room temperature toblock the nonspecific binding of IgG to the proteins. The filter was putin IgG solution prepared by diluting IgG (8.2 mg/ml) affinity purifiedfrom Korean HCV patients with 200-fold volume of PBS containing 0.5%gelatin and 0.05% Tween 20; and mildly shaken for 1 hour at roomtemperature to react the protein and IgG. The filter was then washedwith PBS containing 0.2% Tween 20 for 5 minutes, 4 times. The filter wasput in an anti-human IgG solution prepared by diluting Anti-HumanIgG-HRP labeled with horseradish peroxidase (Bio-Rad Lab., U.S.A., goatanti-human IgG-HRP) with 200-fold volume of PBS containing 0.5% gelatinand 0.05% Tween 20, and shaken for 1 hour at room temperature. Thefilter was washed with PBS containing 0.2% Tween 20 for 5 minutes, 4times; and with 50 mM Tris buffer solution (pH 7.0), 2 times.

To the filter were added 50 mM Tris buffer solution containing 400 μg/ml4-chloro-1-naphthol and 0.03% hydrogen peroxide to develop a colorreaction. The results from the above western blotting are shown in FIG.31-B. In FIG. 31-B, lane 2 shows the result of the extracts of the yeasttransformed with pYLBC-A/G-UB-CORE 14; lane 3 shows the result of theextracts of the yeast transformed with pYLBC-A/G-UB-KHCV 897; lane 5shows the result of the extracts of the yeast transformed withpYLBC-A/G-UB-KHCV 403; lane 6 shows the result of the extracts of theyeast transformed with pYLBC-A/G-UB-KHCV 573; lanes 1 and 4 show theresults of the extracts of yeasts having no KHCV expression vector; andlane M represents the standard protein molecular size markers (unit:kilodalton).

FIG. 32 shows the SPS-PAGE and western blotting results to confirm theproductions of E2N and E2C proteins. In FIG. 32, lane 1 shows theextracts of yeast transformed with a plasmid without KHCV gene; lane 2shows the extracts of the yeast transformed with pYLBC-A/G-UB-E2N; lanes3 to 5 show the extracts of the yeast transformed with pYLBC-A/G-UB-E2N;and lane 6 shows the standard molecular size markers, i.e., 200, 97, 72,43, 29, 18 and 14 kilodaltons from the top of the gel.

EXAMPLE 4 Preparation of the Vector Expressing KHCV cDNA Fragments in E.coli

(4-A): Preparation of Expression Vector Containing trp Promoter

(4-A-1): Preparation of KHCV cDNA Fragments

The fragments K384, K510, K573, K879, E2N and E2C prepared in Example(3-A-1) and (3-A-2) were used.

Envelope 1(E1) fragment, which is located from the 916th to 1509thnucleotides of KHCV-LBC1, was prepared by PCR in the same manner as inExample (3-A-1), using the following primers:

Primer PEIUBI (SEQ ID NO: 56)(5′-CTTGGTGTTGAGACTCCGCGGTGGTTATGAAGTGGGCAACGCGTCC-3′) contains 25nucleotides on the 5′-end region overlapping with ubiquitin gene; regionof the 916th to the 936th nucleotides of KHCV-LBC1.

Primer PEISAL (SEQ ID NO: 57)(5′-GACTGGACTATTACCCTGTCACGTGGGTGGTGGTTCC-3′) contains a codon toterminate translation after the 1509th nucleotide of KHCV-LBC1; and arecognition site of Sal I.

(4-A-2): Preparation of Ubiquitin Gene

<Step 1>

3 different oligonucleotides as disclosed below were designed frominformation on the ubiquitin gene reported by Ozkaynak, et al., EMBO. J.6, 1429-1439(1987) and synthesized using a DNA synthesizer as follows:

UBI1 (SEQ ID NO: 58):5′-CCCCATATGCAAATTTTCGTCAAAACTCTAACAGGGAAGACTATAACCCTAGAGGTTGAATCTTCCGACACTATTGACAACGTCAA-3′

UBI2 (SEQ ID NO: 59):5′-TAGTTGCTTACCAGCAAAAATCAATCTCTGCTGATCCGGAGGGATACCTTCTTTATCTTTGAATTTTACTTTTGACGTTGTCAATAGTCTC-3′

UBI3 (SEQ ID NO: 60):5′-ACCACCGCGGAGTCTCAACACCAAGTGAAGAGTAGATTCCTTTTGGATGTTGTAGTCAGACAAGGTTCTACCATGTTCTAGTTGCTTACCAGCAAAAA-3′

UBI1 was designed to have a recognition site of Nde I (5′-CATATG-3′) atthe 5′-end and about 20 nucleotides overlapping with UBI2; and UBI3 isdesigned to have a recognition site of Sac II (5′-CCGCGG-3′) without anychange in the amino acid sequence encoded therein (see FIG. 33).

<Step 2>

To the mixture of 2 μg of UBI1, 0.02 μg of UBI2 and 2 μg of UBI3 wereadded 10 μl of 10×PCR buffer solution, 10 μl of 2 mM dNTP's mixture and0.5 μl of Taq polymerase; and distilled water was added thereto toadjust the total volume to be 100 μl. The PCR was carried out in thesame manner as in Reference Example 7. The resulting mixture wassubjected to 5% polyacrylamide gel electrophoresis to isolate 240 bp ofDNA, which was named fragment Ub; and the isolated fragment wasdissolved in 20 μl of TE buffer solution.

(4-A-3): Ligation of Ubiquitin Gene to KHCV cDNA

Each of the fragments prepared in Example (4-A-1) was ligated tofragment Ub by PCR as follows.

As primers for the PCR, the primers prepared in Step 1 of Example(3-A-1) and Step 1 of Example (4-A-2) were used.

7 different test tubes were prepared as follows:

Tube A was provided with 50 ng of fragment K384, 50 ng of fragment Ub, 2μg of primer UBIL and 2 μg of primer PSALCORE14; Tube B was providedwith 50 ng of fragment K510, 50 ng of fragment Ub, 2 μg of primer UBI1and 2 μg of primer PSALCORE17; Tube C was provided with 50 ng offragment K573, 50 ng of fragment Ub, 2 μg of primer UBI1 and 2 μg ofprimer PSALCORE22; Tube D was provided with 50 ng of fragment K897, 50ng of fragment Ub, 2 μg of primer UBI1 and 2 μg of primer PKHCV897SAL;Tube E was provided with 50 ng of fragment E2N, 50 ng of fragment Ub, 2μg of primer UBI1 and 2 μg of primer PE2NSAL; Tube F was provided with50 ng of fragment E2C, 50 ng of fragment Ub, 2 μg of primer UBI1 and 2μg of primer PE2CSAL; and Tube G was provided with 50 ng of fragment E1,50 ng of fragment Ub, 2 μg of primer UBI1, and 2 μg of primer PE1SAL.

To each of the tubes were added 10 μl of 10×polymerase reaction buffersolution, 10 μl of 2 mM dNTP's mixture and 0.5 μl of Taq polymerase; anddistilled water was added thereto to adjust the total volume to be 100μl. PCRs were carried out under the same condition as in ReferenceExample 7. Each of the PCR products was digested with NdeI and Sal I inNEB buffer solution 3; and the fragments obtained in Tubes A to G werenamed fragments UBCORE14, UBCORE17, UBCORE22, UBKHCV897, UBE2N, UBE2Cand UBE1, respectively.

(4-A-4): Preparation of the Expression Vector

<Step 1>

2 μg of ptrp 332-HGH (see Korean Patent Publication No. 91-457,KFCC-10667) was completely digested with Pst I and Sal I; and 2 μg ofthe plasmid was completely digested with Pst I and Nde I in NEB buffersolution 4. The products were separated on 0.7% agarose gel from which1.5 Kb and 0.8 Kb fragments were isolated; and named fragments PB andPS, respectively.

<Step 2>

Using the fragments prepared in the above Step 1 and Example (4-A-3),ligation was carried out as follows:

Ligation Tube A was provided with 100 ng of UBCORE14; Ligation Tube Bwas provided with 100 ng of UBCORE17; Ligation Tube C was provided with100 ng of UBCORE22; Ligation Tube D was provided with 100 ng ofUBKHCV897; Ligation Tube E was provided with 100 ng of UBE2N; LigationTube F was provided with 100 ng of UBE2C; and Ligation Tube G wasprovided with 100 ng of UBE1. To each of the tubes were added 100 ng ofPB, 100 ng of PS, 2 μl of 10×ligation buffer solution and 10 units of T4DNA ligase; and distilled water was added thereto to adjust the totalvolume to be 20 μl. The reaction was carried out for 12 hours at 16° C.Each of the ligated vectors was isolated; and E. coli HBL101(ATCC 33694)was transformed with each of the vectors. The vector containing fragmentUBCORE14 was isolated and named ptrpH-UB-CORE14; the vector containingfragment UBCORE17 was isolated and named ptrpH-UB-CORE17; the vectorcontaining fragment UBCORE22 was isolated and named ptrpH-UB-CORE22; thevector containing fragment UBKHCV 897 was isolated and namedptrpH-UB-KHCV897; the vector containing fragment UBE2N was isolated andnamed ptrpH-UB-E2N; the vector containing fragment UBE2C was isolatedand named ptrpH-UB-E2C; and the vector containing fragment UBE1 wasisolated and named ptrpH-UB-E1 (see FIG. 34).

(4-B): Preparation of Vectors pMAL-KHCV Containing tac Promoter

(4-B-1): Amplification of KHCV cDNA Fragments

<Step 1>

In order to express the KHCV cDNA fragments to the MBP-fused proteins inE. coli by employing tac promoter, the primers discribed below weresynthesized using a DNA synthesizer.

Primer PK426R (SEQ ID NO: 61): 5′-CTCCGAATTCGGTGCTTGCGAGTGCCCC-3′

Primer PK426X (SEQ ID NO: 62): 5′-CACGCTCGAGGCATGTGAGGGTGTCGATGAC-3′

Primer PSALCORE17 (SEQ ID NO: 41):5′-GGGGTCGACTATTAGGGCAGATTCCCTGTTGC-3′

Primer P426B (SEQ ID NO: 49): 5′-GGGTGGGCAGGATGGCTCCTG-3′

Primer PK513R (SEQ ID NO: 63):5′-CTCCGAATTCGGCACGAGGCTGGAGGACGGCGTGAACT-3′

Primer PK513X (SEQ ID NO: 64): 5′-CACGCTCGAGAGGCGACCAGTTCATCATCAT-3′

Primer PK80R (SEQ ID NO: 65):5′-CTCCGAATTCGGCACGAGGGTTTCCCAGCTGTTCACCTT-3′

Primer PK810X (SEQ ID NO: 66): 5′-CACGCTCGAGATTCAGCCATGTACAACCGAACC-3′

Primer PK798R (SEQ ID NO: 67):5′-CTCCGAATTCGGCACGAGGGACGTGCTGCTCCTTAAC-3′

Primer PK798X (SEQ ID NO: 68): 5′-CACGCTCGAGCAGAAGCAGCGGCCATACGCC-3′

Primer PK754R (SEQ ID NO: 69):5′-AAAAAGAATTCGGCACGAGGCTGCGAGATTGGGCTCACACG-3′

Primer PK754X (SEQ ID NO: 70):5′-AAAAACTCGAGCCGCATAGTAGTTTCCATAGACTCAACGGGTATGAATT-3′

Primer PK652R (SEQ ID NO: 71):5′-AAAAAGAATTCGGCACGAGGTTCATACCCGTTGAGTCTATGGAA-3′

Primer PK652X (SEQ ID NO: 72):5′-ATTATTGTCGACTATCTATCTACTCGAGTCACAGCTTTGCAGCGAGCTCGT-3′

Primer PK403R (SEQ ID NO: 73): 5′-AAAAAGAATTCACGGGCATGACCACTGAC-3′

Primer PK403X (SEQ ID NO: 74): 5′-ATTATTCTCGAGTATCACTCTTGCCGCCACAAGAG-3′

Primer PK271R (SEQ ID NO: 75): 5′-AAAAAGAATTCACTAGCCTTACAGGCCGG-3′

Primer PK271X (SEQ ID NO: 76): 5′-CACGCTCGAGTCACGTGACCAGGTAAAGGTC-3′

Primer PK495R (SEQ ID NO: 77):5′-CCCCCGAATTCGGCACGAGCGCTGCGGAGGAAAGCAAGTT-3′

Primer PK495X (SEQ ID NO: 78): 5′-AAAAACTCGAGGACCACGTCATAAAGGGCCA-3′

Primer PK494R (SEQ ID NO: 79):5′-AAAAGAATTCGGCACGAGCGATGCATCTGGTAAAAGGGT-3′

Primer PK494X (SEQ ID NO: 80): 5′-AAAACTCGAGATTGGAGTGAGTTTGAGCTT-3′

<Step 2>

11 different test tubes were prepared, which were provided with theprimers as follows:

Tube A: Primer PK426R 2 μg, Primer PK426X 2 μg

Tube B: Primer PK426R 2 μg, Primer PK426B 20 ng, PSALCORE17 20 μg

Tube C: Primer PK513R 2 μg, Primer PK513X 2 μg

Tube D: Primer PK810R 2 μg, Primer PK810X 2 μg

Tube E: Primer PK798R 2 μg, Primer PK798X 2 μg

Tube F: Primer PK754R 2 μg, Primer PK754X 2 μg

Tube G: Primer PK652R 2 μg, Primer PK652X 2 μg

Tube H: Primer PK403R 2 μg, Primer PK403X 2 μg

Tube I: Primer PK271R 2 μg, Primer PK271X 2 μg

Tube J: Primer PK495R 2 μg, Primer PK495X 2 μg

Tube K: Primer PK494R 2 μg, Primer PK494X 2 μg

To each of the tubes were added 10 ng of KHCV-LBC1(ATCC 75008), 10 μl of10×polymerase buffer solution, 10 μl of 10 mM dNTP's mixture and 0.5 μl(2 units) of Taq polymerase; and distilled water was added thereto toadjust the total volume to be 100 μl.

Each of the reaction mixtures was added 50 μl of mineral oil to preventevaporation; and PCRs were carried out in the same manner as inReference Example 7.

(4-B-2): Preparation of Expression Vector

2 μg of pMAL-CR1 (New England Biolabs Inc., Cat. No. 800, 11099 NorthTorrey Pines Road, La Jolla, Calif., U.S.A.) was completely digestedwith Eco RI and Sal I in NEB buffer solution 3. The product was exractedwith phenol/chloroform and precipitated with ethanol. The precipitatewas dissolved in 40 μl of TE buffer solution.

The PCR products prepared in Step 2 of Example (4-B-1) were digestedwith Eco RI and Xho I as follows:

1 μl of each of the PCR products in Tubes A and C to F, H and J to M wascompletely digested with Eco RI and Xho I; 3 μg of each of the PCRproducts in Tubes G and I was completely digested with Xho I and thenpartially digested with Eco RI; and 1 μl of the PCR product in tube Cwas completely digested with Eco RI and Sal I. Eco RI-Xho I and EcoRI-Sal I fragments so obtained were isolated and dissolved in 20 μl ofTE buffer solution in the same manner as in Reference Example 1,respectively.

To 5 μl of each of the above cDNA fragments digested with Eco RI-Xho Iand Eco RI and Sal I were added 2 μl of 10×ligation buffer solution, 1μl (50 ng) of pMal-CR1 treated with Eco RI and Sal I above and 10 unitsof T4 DNA ligase; and distilled water was added thereto to adjust thetotal volume to be 20 μl. The reaction was carried out for 12 hours at16° C.

Each of the ligated vectors was isolated; and E. coli HB101(ATCC 33694)was transformed with each of the recombinant vectors. The vectors inTubes A to K were named pMAL-KHCV426, pMAL-KHCV555, pMAL-KHCV513,pMAL-KHCV810, pMAL-KHCV798, pMAL-KHCV754, pMAL-KHCV652, pMAL-KHCV403,pMAL-KHCV271, pMAL-KHCV495 and pMAL-KHCV494, respectively.

The vector used for the above recombinant vector, pMAL-CR1, is describedin FIG. 35.

(4-C): Expression of KHCV cDNA Fragments in E. coli

(4-C-1): Expression of KHCV cDNA Fragments by Vector Containing trpPromoter

<Step 1>

E. coli W3110(ATCC 38335) was transformed with each of the plasmidsprepared in Example (4-A). Of them, E. coli W3110 transformed withptrpH-UB-KHCV897 (E. coli W3110 ptrpH-UB-KHCV 897) was deposited withthe accession number of ATCC 69640 on Jun. 27, 1991; E. coli W3110transformed with ptrpH-UB-CORE17 (E. coli W3110 ptrpH-UB-CORE 17) wasdeposited with the accession number of ATCC 68641 on Jun. 27, 1991; E.coli W3110 transformed with ptrpH-UB-CORE14 (E. coli W3110 ptrpH-UB-CORE14) was deposited with the accession number of ATCC 68642 on Jul. 1,1991; E. coli W3110 transformed with ptrpH-UB-E1 (E. coli W3110ptrpH-UB-E 1) was deposited with the accession number of ATCC 68878 onDec. 11, 1991; and E. coli W3110 transformed with ptrpH-UB-E2N (E. coliW3110 ptrpH-UB-E2N) was deposited with the accession number of ATCC68966 on Apr. 22, 1992, to American Type Cultue Collection under theterms of Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure.

E. coli transformed with ptrpH-UB-CORE14 was cultured with shaking inliquid LB medium (1% Bacto-tryptone, 0.5% yeast extracts, 1% NaCl)containing 50 μg/ml ampicillin at 37° C. for 12 hours. 5 ml of theculture was transferred into 1 l of M9 medium (40 mM K₂HPO₄, 22 mMKH₂PO₄, 8.5 mM NaCl, 18.7 mM NH₄Cl, 1% glucose, 0.1 mM MgSO₄, 0.1 mMCaCl₂, 0.4% casamino acid, 10 μl/ml Vit. B₁, 40 μg/ml ampicillin); andcultured with shaking for 3 to 4 hours at 37° C. When its O.D. value at650 nm reached 0.5, indolacrylic acid (IAA) was added to the culture toadjust the final concentration to be 1.4 mM. After 5 hours, theresulting culture was centrifuged at 3000 rpm for 25 minutes to collectthe E. coli cell precipitate.

The other recombinant E. coli cells were cultured in the same manner asabove to produce the KHCV proteins.

<Step 2>

Each of the cells was suspended in the buffer solution and thensubjected to 15% SDS-PAGE by employing Laemmli's method (Nature 227,680(1970)) to confirm the expression of the ubiquitin-KHCV protein. Theresults are shown in FIGS. 36 to 38.

In FIG. 36, lane M represents the standard molecular size marker, i.e.,72, 43, 29 and 18 kilodaltons from the top; lane 1 shows the products ofE. coli having plasmid without KHCV gene; lane 2 shows the products ofE. coli transformed with ptrpH-UB-CORE14 wherein 23 kd protein wasproduced; lane 3 shows the products of E. coli transformed withptrpH-UB-CORE17 wherein 27 kd protein was produced; lane 4 shows theproducts of E. coli transformed with ptrpH-UB-CORE22 wherein 29 kdprotein was produced; lane 5 shows the products of E. coli transformedwith ptrpH-UB-KHCV897 wherein 40 kd protein was produced; and lane 6shows the purified KHCV UB 897, protein.

In FIG. 37, lane 1 shows the products of E. coli having plamid withoutKHCV gene; lanes 2 to 5 show the products of E. coli transformed withptrpH-UB-E1 harvested after 2,4,6 and 12 hours from the addition time ofIAA, respectively; and lane 6 represents the standard molecular sizemarkers, i.e., 72, 43, 29, 18 and 14 kilodaltons from the top.

In FIG. 38, lane 1 shows the products of E. coli having plasmid withoutKHCV gene; lane 2 shows the products of E. coli transformed withptrpH-UB-E2C; and lane 3 shows the products of E. coli transformed withptrpH-UB-E2N.

Western blotting was carried out in the same manner as in Example (3-C)to confirm that the proteins produced in recombinant E. coli arespecifically bound to KHCV antibody. The results are shown in FIGS. 39to 41.

(4-C-2): Expression of KHCV cDNA by Vector Containing tac Promoter

<Step 1>

E. coli D1210(ATCC 27325) was transformed with each of the plasmidsprepared in Example (4-B) in the same manner as in Reference Example 4.Among them, E. coli D1210 transformed with pMAL-KHCV555 (E. coli D1210pMAL-KHCV555) was deposited with the accession number of 68639 on Jun.27, 1991 at American Type Cultrue Collection under the terms of BuadpestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure.

The transformed E. coli was cultured in liquid LB medium containing 50μl/ml ampicillin with shaking for 12 hours, and 5 ml of the culture wastransferred into 1 l of M9 medium (6 g of Na₂HPO₄, 3 g of KH₂PO₄, 0.5 gof NaCl, 1 g of NH₄Cl, 2 μl of 1M MgSO₄, 100 μl of 20% glucose, 0.1 mlof CaCl₂ per liter) and cultured with shaking for 3 to 4 hours at 37° C.When its O.D. value at 650 nm reached 0.5, IPTG was added to the culutreto adjust its concentration to be 0.2 mM. After 5 hours, the resultingculture was centrifuged at 3000 rpm for 25 minutes to collect the E.coli cell precipitate.

<Step 2>

The cell precipitate was suspended in a buffer solution and thensubjected to 15% SDS-PAGE by employing Laemmli's method (Nature 227,680(1970)) to confirm the expression of KHCV proteins. The results areshown in FIG. 42. In FIG. 42, lane M represents the standard molecularsize marker; lane 1 shows the products of E. coli transformed withpMAL-CR1, wherein 40 kd protein was produced; lane 2 shows the productsof E. coli transformed with pMAL-KHCV 426, wherein 65 kd protein(MBP-KHCV 426 protein) was produced; lane 3 shows the products of E.coli transformed with pMAL-KHCV 555, wherein 70 kd protein (MBP-KHCV555protein) was produced; lane 4 shows the products of E. coli transformedwith pMAL-KHCV513, wherein 65 kd protein (MBP-KHCV513 protein) wasproduced; lane 5 shows the products of E. coli transformed withpMAL-KHCV810, wherein 75 kd protein (MBP-KHCV810 protein) was produced;lane 6 shows the products of E. coli transformed with pMAL-KHCV798,wherein 72 kd protein (MBP-KHCV798 protein) was produced; lane 7 showsthe products of E. coli transformed with pMAL-KHCV27, wherein 50 kdprotein (MBP-KHCV271 protein) was produced; lane 8 shows the products ofE. coli transformed with pMAL-KHCV754, wherein 72 kd protein(MBP-KHCV754 protein) was produced; lane 9 shows the products of E. colitransformed with pMAL-KHCV652, wherein 70 kd protein (MBP-KHCV652protein) was produced; lane 10 shows the products of E. coli transformedwith pMAL-KHCV403, wherein 65 kd protein (MBP-KHCV403 protein) wasproduced; lane 11 shows the products of E. coli transformed withpMAL-KHCV495, wherein 70 kd protein (MBP-KHCV495 protein) was produced;lane 12 shows the products of E. coli transformed with pMAL-KHCV494,wherein 70 kd protein (MBP-KHCV494 protein) was produced.

Western blotting was carried out in the same manner as in Example (3-C)to confirm that the above proteins are specifically bound to KHCVantibody. The results are shown in FIG. 43.

(4-C-3): Digestion of MBP from Fused Protein

Each of the MBP-fused proteins was dialysed to Factor Xa buffer solution(20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM CaCl₂, 1 mM azide) for 24hours. 0.2 μg of each of the dialysed proteins (1 mg/ml) was then mixedwith 0.2 μg of Factor Xa (New England Biolabs Inc., Cat. #800-10L); andthe reaction mixtures were stood for 24 hours at room temperature.

Each of the resulting mixtures was heated to 100° C. for 5 minutes; andthe products were subjected to SDS-PAGE in the same manner as in Example(1-C) to confirm that the MBPs were removed from their fused proteins.The MBP-removed proteins were named KHCV 426 protein, KHCV 555 protein,KHCV 513 protein, KHCV 810 protein, KHCV 798 protein, KHCV 271 protein,KHCV 754 protein, KHCV 652 protein, KHCV 403 protein, KHCV 495 proteinand KHCV 494 protein, respectively.

As described above, various lengths and sequences of KHCV cDNAs for thepreparation of expression vectors could be prepared by PCR method usingvarious combinations of the primers; and, therefore, it is apparent thatother similar KHCV cDNA fragments can be readily synthesized by oneskilled in the art, on the basis of the above disclosures. It is alsoapparent that other KHCV antigen proteins can be readily synthesized byone skilled in the art on the basis of the above disclosures since suchKHCV antigen proteins are dependent on the KHCV cDNA. Further, it isapperant that, for the preparation of KHCV cDNAs and KHCV antigenproteins, not only the enzymes, linkers and the other materials used inExamples but also their equivalents can be employed.

EXAMPLE 5 Purification of KHCV Protein Expressed in Yeast Cell

(5-A): Purification of KHCV 403 Protein

Step 1: Culture of Recombinant Yeast Cell

Saccharomyces cerevisiae DCO4-UB-KHCV403 transformed with a vector(pYLBC-A/G-UB-KHCV403) containing KHCV 403 cDNA fragment and ubiquitingene was cultured in 10 ml of a leucine-deficient medium (0.67% yeastnitrogen base without amino acid, 5% glucose and 0.25% of mixture ofamino acids without leucine) at 30° C. for 12 hours; then, the culturewas transferred into 100 ml of YEPD medium containing 5% glucose (2%peptone, 1% yeast extract, 5% glucose) and cultured with shaking at atemperature of 30° C. for about 6 hours; and the culture was transferredto 1 l of YEPD medium containing 5% glucose and cultured at 30° C. for 6hours, to obtain a seed culture for fermentation.

10 l of YEPD medium containing 2% glucose was charged to 14 l fermentor(Bench Top Fermentor: NBS Company, U.S.A.); and the seed culture wasinoculated thereto and cultured with shaking at a speed of 250 rpm andat 30° C. for about 48 hours. The culture was centrifuged at a speed of2500 rpm for 20 minutes with a centrifuge (Beckman J-6B, Rotor JS 4.2)to obtain the recombinant yeast cell paste.

Step 2: Disruption of Yeast Cells

The recombinant yeast cell obtained in Step 1 was suspended in 500 ml ofbuffer (50 mM Tris, pH 8.5, 5 mM EDTA, 10 mM β-mercaptoethanol, 1 mMphenylmethylsulfonylfluoride, 1 μg/ml pepstatin A); and glass beadshaving a diameter of 0.4 mm were added in an amount equivalent to 50%(v/v) of the total volume. The resultants were homogenized at 4° C. for5 minutes with a homogenizer (Bead Beater, Biospec Product, U.S.A.) todisrupt the cell membrane. The disrupted cells were filtered using afilter (Whatman, 3MM, U.S.A.) to remove the glass beads and obtain theyeast homogenate.

Step 3: Identification of Specific Antigen Protein

A small amount of the yeast homogenate obtained in Step 2 was subjectedto electrophoresis on 15% SDS-polyacrylamide gel. The result showed thatubiquitins were excised in the cell and proteins expressed from KHCV 403cDNA (hereinafter referred to as KHCV 403 protein) were produced with amolecular weight of about 17,000 dalton.

The proteins separated on the gel were blotted onto a nitrocellulosefilter; and then the filter was placed into phosphate buffered saline(PBS: 10 mM phosphate, 0.15M NaCl, pH 7.0) containing 0.5% Tween-20 in atray and mildly stirred at a room temperature for 2 hours to blocknon-specific binding of immunoglobulin G. Subsequently, immunoglobulin G(8.2 mg/ml) which was affinity purified from the serum of a patient withKorean hepatitis C was diluted in a ratio of 1/200 (v/v) with PBScontaining 0.5% gelatin and 0.05% Tween 20; 10 ml of the diluted IgG wasadded to the filter; the tray was shaken mildly at a room temperaturefor 1 hour; and the filter was washed four times for 5 minutes each withPBS containing 0.05% Tween-20. An anti-human immunoglobulin G labelledwith horseradish peroxidase (Bio Rad Lab, Goat Anti-Human IgG-HRP) wasdiluted with PBS containing 0.5% gelatin and 0.05% Tween-20 in a ratioof 1/200 (V/V) and added to the filter. The filter was reacted with mildshaking at a room temperature for 1 hour. The filter was washed fourtimes for 5 minutes each with PBS containing 0.05% Tween-20 and thentwice with 50 mM Tris buffer (pH 7.0). To the filter was added 50 mMTris buffer (pH 7.0) containing 400 μg/ml 4-chloro-1-naphtol and 0.03%hydrogen peroxide to develop a color reaction. The result showed thatKHCV 403 protein of an entire yeast homogenate alone was immunologicallyreacted with the serum of the patient with hepatitis C to exhibit avisible band; and, therefore, said KHCV 403 protein alone is animmunoreactive protein which can bind to antibodies against KHCV.

Step 4: Removal of Dissolved Protein

The yeast homogenate obtained in Step 2 was centrifuged at 11,000 rpmwith a centrifuge (Beckmn J2-21, Rotor JA 14) to remove the supernatantand obtain the insoluble precipitate containing KHCV 403 protein.

Step 5: Dissolution and Fractionation of the Precipitate with Urea

The precipitate obtained in Step 4 was dissolved in 750 ml of a buffer(50 mM Tris, pH 8.5, 5 mM EDTA, 10 mM β-mercaptoethanol, 1 mMphenylmethylsulfonylfluoride, 1 μg/ml pepstatin A) containing 8M urea.The solution was centrifuged to remove undissolved precipitates andcollect the supernatant. The supernatant was dialyzed with a buffer (10mM Tris, pH 9.0, 2 mM EDTA, 5 mM β-mercaptoethanol) containing 2M ureaand centrifuged to remove the precipitates and obtain the supernatantcontaining KHCV 403 protein.

Step 6: First DEAE Ion Exchange Chromatography

The supernatant obtained in Step 5 was passed over DEAE-Sepharose column(Pharmacia, FF, 5 cm×15 cm, U.S.A.) equilibrated with a buffer (10 mMTris, pH 9.0, 2 mM EDTA, 5 mM β-mercaptoethanol) containing 2M urea. Thebound proteins were eluted by adding 750 ml of a buffer (10 mM Tris, pH9.0, 2 mM EDTA, 5 mM β-mercaptoethanol) containing 0.2M sodium chloride.

Step 7: Second DEAE Ion Exchange Chromatography

The protein fractions which contained KHCV 403 protein were collectedand dialyzed with a buffer (10 mM Tris, pH 9.0, 2 mM EDTA, 5 mMβ-mercaptoethanol) to remove urea and then passed over DEAE-Sepharosecolumn equilibrated with said buffer. A buffer (10 mM Tris, pH 9.0, 2 mMEDTA, 4 mM β-mercaptoethanol) containing 0.1M sodium chloride was addedto separate out the eluted protein; and 500 ml of the buffer having aconcentration gradient of 0.1M to 0.2M sodium chloride was added tofractionate the column-bound proteins. The fractions were subjected toSDS-PAGE to collect the fractions containing highly purified KHCV 403protein.

Step 8: FPLC-phenyl Chromatography

The fractions obtained in Step 7 were dialyzed with a buffer (50 mMTris, pH 7.4, 2 mM EDTA, 5 mM β-mercaptoethanol) containing 1.5M sodiumchloride and passed over FPLC-phenyl superose column (Pharmacia, HR10/10, 1 cm×8 cm, U.S.A.) equilibrated with said buffer; and 160 ml ofthe buffer containing a concentration gradient of 1.5 to 0M sodiumchloride was added to fractionate the proteins. The fractions weresubjected to SDS-PAGE to identify the purity. The fractions containinghighly purified KHCV 403 proteins were separately pooled to obtain KHCV403 proteins having a purity of more than 95%.

(5-B): Purification of KHCV CORE 14 Protein

Step 1: Culture of Recombinant Yeast Cells

Saccharomyces cerevisiae DCO4-UB-CORE 14 transformed with a vector(pYLBC-A/G-UB-CORE 14) containing a cDNA fragment encoding KHCV CORE 14protein and ubiquitin gene was cultured in a leucine deficient mediumcontaining 5% glucose in accordance with the process of Step 1 ofExample (5-A); 20 ml of the culture was transferred to 100 ml of YEPDmedium containing 4% glucose and cultured with shaking at 30° C. for 6hours; and transferred to 1 l of YEPD medium containing 2% glucose andcultured at 30° C. for 24 to 48 hours. The culture was centrifuged tocollect cell precipitates.

Step 2: Disruption of Yeast Cells

The recombinant yeast cell precipitates obtained in Step 1 weresuspended in 30 ml of a buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 10 mMβ-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride, 1 μg/mlpepstatin); and glass beads having a diameter of 0.4 mm were added in anamount equivalent to 50% of the total volume. The resultants werehomogenized for 5 minutes at 4° C. with a homogenizer (Bead Beater,Biospec Product, U.S.A.) 3 times to disrupt the cell membrane and obtainthe yeast homogenate.

Step 3: Identification of Specific Antigen Protein

A small amount of the yeast homogenate obtained in Step 2 was subjectedto electrophoresis on 15% SDS-polyacrylamide gel and stained withcoomassie brilliant blue. The result showed that the ubiquitin wasexcised from the KHCV protein and the protein expressed in KHCV cDNA(hereinafter referred to as KHCV CORE 14 protein) was produced with amolecular weight of about 16,000 dalton.

Western blotting was carried out in accordance with Step 3 of Example(5-A). The result indicated that KHCV CORE 14 protein alone wasimmunologically reactive with the serum of the patient with hepatitis Cto exhibit a visible band.

Step 4: Removal of Soluble Proteins and Washing of Insoluble Precipitate

The yeast homogenate obtained in Step 2 was centrifuged at 11,000 rpmwith a centrifuge (Beckman J2-21, Rotor JA-14) to remove dissolvedproteins and obtain insoluble precipitate containing KHCV CORE 14protein. The precipitate was suspended in 0.5 l of PBS containing 1%Triton X-100, 1 mM EDTA and 10 mM β-mercaptoethanol with stirring for 10minutes and centrifuged. The precipitate was washed once with 10 mMphosphate solution (pH 6.5).

Step 5: Dissolution of the Precipitate with 8M Urea

The insoluble precipitate obtained in Step 4 was suspended in 10 mMsodium phosphate solution (pH 6.5) containing 8M urea, 1 mM EDTA and 10mM β-mercaptoethanol; and stirred for 12 hours at 4° C. to dissolve KHCVCORE 14 protein. The solution was centrifuged for 20 minutes at 15,000rpm with a centrifuge (Beckman J2-21, Rotor JA20) to obtain thesupernatant.

Step 6: CM-ion Exchange Resin Chromatography

The solution containing KHCV CORE14 protein obtained in Step 5 waspassed at a flow rate of 1 ml/min. over a column (2.5 cm×10 cm) having25 ml of CM (carboxymethyl)-Sepharose resin (Pharmacia, Sweden)equiribrated with a buffer (pH6.5) containing 6M urea, 1 mM EDTA, 10 mMβ-mercaptoethanol and 10 mM phosphate. The materials remaining in thecolumn in free form were thoroughly washed with said equilibratingbuffer solution. The proteins adsorbed in the column were eluted at aflow rate of 3 ml/min. with 500 ml of said equilibrating buffer solutionwith a concentration gradient of 0 to 0.5M sodium chloride. The eluatewas subjected to SDS polyacrylamide gel electrophoresis, which indicatedthat KHCV CORE14 protein was eluted at about 0.3M sodium chloride.Fractions containing KHCV CORE 14 protein were collected for use in thenext step.

Step 7: S-200 Gel Permeation Chromatography

The fractions collected in Step 6 were passed over YM5 ultrafiltrationmembrane (Amicon, U.S.A.) to concentrate to 10 ml. The concentrate waspassed over S-200 Sephacryl column (Pharmacia, Sweden, 2.5 cm×100 cm)equilibrated with PBS solution containing 6M urea, 1 mM EDTA and 10 mMβ-mercaptoethanol at a flow rate of 0.5 ml/min. to separate themaccording to the in molecular weight. The collected protein fractionswere subjected to 15% SDS-polyacrylamide gel electrophoresis. Fractionscontaining highly purified KHCV CORE14 protein were collected anddialyzed with PBS buffer at 4° C. to remove urea and obtain 4 mg ofhighly purified KHCV CORE14 protein.

It should be understood that the proteins encoded in other KHCV cDNAfragments expressed in yeast may also be purified by other processessimilar to one described above.

EXAMPLE 6 Purification of KHCV Protein Expressed in E. coli

(6-A): Purification of KHCV UB 897 Protein

Step 1: Culture of Recombinant E. coli

E. coli W3110 ptrpH-KHCV 897(ATCC 68640) transformed with a vector(ptrpH-UB-KHCV 897) comprising KHCV 897 cDNA fragment with ubiquitingene was cultured with shaking for 12 hours in LB medium (10 g ofBactotriptone, 5 g of yeast extract, 10 g of NaCl per liter) containing50 μg/ml of ampicillin. 5 ml of the culture was transferred to 1 l of M9medium (40 mM K₂HPO₄, 22 mM KH₂PO₄, 8.5 mM NaCl, 18.7 mM NH₄Cl, 1%glucose, 0.1 mM MgSO₄, 0.1 mM CaCl₂, 0.4% casamino acid, 10 μg/ml ofVit. B₁) containing 40 μg/ml of ampicillin and cultured with shaking forabout 3 to 4 hours at 37° C. Indoleacrylic acid (IAA) was added so as tomake the final concentration of 0.14 mM and produce KHCV UB 897 proteinwhen O.D. value of the culture at 650 nm reached 0.5. After about 5hours from the addition of IAA, the cell culture was centrifuged at2,500 rpm for 20 minutes with a centrifuge (Beckman J-6B, Rotor JS 4.2)to obtain E.coli cell precipitate. The precipitate was washed once withphosphate buffered saline (10 mM phosphate, pH 7.0, 0.15M sodiumchloride).

Step 2: Disruption of Cells

3 g of E. coli cell precipitate obtained in Step 1 was suspended in 40ml of a buffer (50 mM Tris, pH 8.5, 5 mM EDTA, 2 mM β-mercaptoethanol, 1mM phenylmethylsulfonylfluoride, 1 μg/ml pepstatin A). 0.3 ml of 50mg/ml lysozyme solution was added to the suspension, left at 37° C. for1 hour and subjected to ultrasonication on ice for 5 minutes and at anoutput of 70% with an ultrasonicator (HEAT SYSTEMS-ULTRASONICS INC.,W225, U.S.A.) to disrupt the cell and obtain a homogenate of E. colicell.

Step 3: Identification of Specific Antigen Protein

A small amount of the homogenate of E. coli cell obtained in step 2 wassubjected to 12% SDS-PAGE. The result indicated that the KHCV proteinexpressed by said vector (hereinafter referred to as KHCV UB 897protein) have a molecular weight of 39,000 dalton.

Thereafter, proteins separated on gel were transferred onto anitrocellulose filter and subjected to Western-blotting in the samemanner as Step 3 of Example (5-A). The result showed that only KHCV UB897 protein was immunologically reacted with the serum of the patientwith hepatitis C to exhibit a visible band. In the light of the result,it can be seen that said expressed KHCV UB 897 protein is animmunoreactive protein which can bind to antibodies against HCV.

Step 4: Removal of Soluble Protein

The cell homogenate obtained in Step 2 was centrifuged at 11,000 rpm for25 minutes with a centrifuge (Beckman J2-21, Rotor JA 14) to removedissolved proteins and obtain insoluble precipitate.

Step 5: Washing of Insoluble Precipitate with Triton X-100 and TrisBuffer

The precipitate obtained in Step 4 was suspended in 50 ml of a buffer(50 mM Tris, pH 8.5, 5 mM EDTA, 2 mM β-mercaptoethanol) containing 1%Triton X-100. The suspension was stirred at a room temperature for 30minutes and centrifuged at 11,000 rpm for 25 minutes with a centrifuge(Beckman J2-21, Rotor JA 14) to remove the supernatant and obtaininsoluble precipitate. Subsequently, the precipitate was suspended in 50ml of a buffer (50 mM Tris, pH 8.5, 5 mM EDTA, 2 mM β-mercaptoethanol).The suspension was stirred and recentrifuged to remove the supernatantand obtain insoluble precipitate.

KHCV UB 897 protein having a purity of at least 60% was obtained throughthe above simple washing procedure only.

Step 6: Dissolution of Insoluble Precipitate with 8M Urea

The insoluble precipitate containing KHCV UB 897 protein obtained inStep 5 was suspended in 50 ml of a buffer containing 8M urea (20 mMphosphate, pH 6.0, 2 mM EDTA, 2 mM β-mercaptoethanol). The suspensionwas stirred at a room temperature for 1 hour and centrifuged to removeinsoluble precipitate and obtain the supernatant.

Step 7: S-Sepharose Ion Exchange Chromatography

The supernatant obtained in Step 6 was passed over S-Sepharose column(Pharmacia, FF, 2.5 cm×7 cm, U.S.A.) equilibrated with a buffer (20 mMphosphate, pH 6.0, 2 mM EDTA, 2 mM β-mercaptoethanol) containing 4M ureaand was eluted with 600 ml of the buffer having a concentration gradientof 0 to 0.2M sodium chloride. Protein fractions were subjected toSDS-PAGE to collect the fractions comprising highly purified KHCV UB 897protein.

Step 8: Removal of Urea and FPLC-Mono Q Ion Exchange Chromatography

The protein fractions comprising KHCV UB 897 protein collected in Step 7were dialyzed against a buffer (10 mM Tris, pH 8.5, 2 mM EDTA, 2 mMβ-mercaptoethanol) to remove urea, loaded over FPLC-Mono Q ion exchangeresin column (Pharmacia, HR 5/5) equilibrated with said buffer andeluted with 40 ml of the buffer having a concentration gradient of 0 to0.4M sodium chloride. The fractions comprising high purified KHCV UB 897protein were collected to obtain KHCV UB 897 protein having a purity ofat least 90%.

(6-B): Purification of KHCV UB CORE 17 Protein

Step 1: Culture of Recombinant E. coli

E. coli W3110 ptrpH-UB-CORE 17(ATCC 68641) transformed with a vactor(ptrpH-UB-CORE 17) containing a cDNA of hepatitis C virus and ubiquitingene was cultured in LB medium containing 50 μg/ml ampicillin, 100 μg/mltryptophan at 37° C. for 12 hours; 50 ml of the culture was transferedto 1 l M9 medium and cultured at 37° C. for 6 to 8 hours; and collecteda cell precipitate as described in step 1 of Example (6-A).

Step 2: Disruption of Cell

3 g of E. coli cell precipitate obtained in Step 1 was suspended in 20ml of a buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 10 mM β-mercaptoethanol,1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin) at 4° C. 3 mg oflysozyme was added to the suspension and stirred for 5 minutes. Theresultant was subjected to ultrasonic treatment for 20 minutes in an icebath with an ultrasonicator (Heat Systemas-Ultrasonics, Inc., W225,U.S.A.) to disrupt the cells and obtain a cell homogenate.

Step 3: Identification of Specific Antigen Protein

Said E. coli cell homogenate obtained in Step 2 was subjected toelectrophoresis on 15% SDS-polyacrylamide gel and stained with coomassiebrilliant blue. The result indicated that the protein having a molecularweight of about 27,000 dalton (hereinafter referred to as KHCV UB CORE17 protein) was produced.

Subsequently, proteins separated on gel were transferred onto anitrocellulose filter, which was subjected to western-blotting inaccordance with the same process as in Step 3 of Example (5-A). Theresult showed that only KHCV UB-CORE 17 protein in the whole E. colicell homogenate was immunologically reacted with the serum of thepatient with hepatitis C to exhibit-a visible band.

Step 4: Treatment with Urea

The cell homogenate obtained in Step 2 was centrifuged at 12,000 rpm for20 minutes with a centrifuge (Beckman J2-21, Rotor JA2) to removeinsoluble materials and obtain the supernatant. To the supernatant wasadded 9M urea solution to a final concentration of 6M and stirred at 4°C. for 12 hours.

Step 5: Treatment with Acid

To the solution obtained in Step 4 was added 1M sodium acetate (pH 4.5)to a concentration of 10 mM; and 1M acetic acid to pH 5.0. The mixturewas stirred for 1 hour and centrifuged at 11,000 rpm with a centrifuge(Beckman J2-21, Rotor JA 14) to remove the precipitate and obtain thesupernatant.

Step 6: Mono-S Chromatography

The supernatant obtained in Step 5 was purified by passing it over FPLCMono-S column (HR 5/5, Pharmacia, Sweden). UB-CORE 17 protein solutionwas loaded over the column equilibrated with buffer A (pH 5.0)containing 8M urea, 1 mM EDTA, 1 mM β-mercaptoethanol and 10 mM aceticacid, which was then washed with said buffer A. Thereafter, buffer Bcontaining 8M urea, 1 mM EDTA, 1 mM β-mercaptoethanol, 10 mM acetic acidand 1M sodium chloride was added gradually to an amount of 17.5% forfirst 5 minutes, 35% for next 55 minutes and 100% for final 10 minutesat a flow rate of 0.8 ml/min to elute the protein. KHCV UB-CORE 17protein was eluted when the amount of buffer B reached 25%, i.e., whenthe concentration of sodium chloride was 0.25M.

Step 7: S-200 Gel Permeation Chromatography

The protein solution obtained in Step 6 was passed over S-200 Sephacrylcolumn (Pharmacia, Sweden, 2.5 cm×100 cm) equilibrated with PBS solutioncontaining 6M urea, 1 mM EDTA and 1 mM β-mercaptoethanol at a flow rateof 0.5 ml/min. to separate it according to the molecular weight. Proteinfractions were collected and subjected to SDS-polyacrylamide gelelectrophoresis to collect the fractions comprising KHCV UB-CORE 17protein. The fractions were dialyzed against PBS solution at 4° C. toobtain 4 mg of KHCV UB-CORE 17 protein having a purity of at least 90%.

(6-C): Purificiation of UB-E1 Protein

Step 1: Culture of Recombinant Bacterial Cell

E. coli W3110 ptrpH-UB-E1(ATCC 68878), which is capable of producing afused protein of KHCV E1 protein and ubiquitin (UB), was cultured andcollected in accordance with the same process as in Step 1 of Example(6-A).

Step 2: Disruption of Cell

The bacteria cell precipitate obtained in Step 1 was suspended in 50 mlof a buffer 1 (20 mM Tris, pH 7.5, 1 mM EDTA, 2 mM β-mercaptoethanol, 1mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A). A lysozymesolution was added to the suspension to a final concentration of 0.2mg/ml, cultured at 37° C. for 30 minutes and subjected to ultrasonictreatment on ice at an output of 70% and for 5 minutes with anultrasonicator to disrupt the cells and obtain the homogenate.

Step 3: Identification of Expression of Specific Antigen

The homgenate obtained in Step 2 was subjected to electrophoresis on 15%SDS-polyacrylamide gel, which indicated that proteins having a molecularweight of about 27,000 dalton (hereinafter referred to as UB-E1 protein)were expressed with the vector.

The proteins separated on gel were blotted onto Immobilon P filter(MILLIPORE, Cat. No. IPUH 00010, pore size 0.45 μm) and subjected towestern-blotting in the same manner as in Step 3 of Example (5-A).

The result showed that only UB-E1 protein in the entire cell homogenatewas immunologically reacted with the serum of the patient with hepatitisC to produce a visible band.

Step 4: Removal of Soluble Protein

The cell homogenate obtained in Step 2 was centrifuged at 11,000 rpm for25 minutes with a centrifuge (Beckman J2-21, Rotor JA14) to removesoluble proteins and obtain insoluble precipitate.

Step 5: Washing of Insoluble Precipitate

The precipitate obtained in Step 4 was suspended in 30 ml of a buffer 1(20 mM Tris, pH 7.5, 1 mM EDTA, 2 mM β-mercaptoethanol) containing 1%Triton X-100. The suspension was stirred at a room temperature for 30minutes and centrifuged at 11,000 rpm for 25 minutes with a centrifuge(Beckman J2-21, Rotor JA 14) to remove proteins soluble in 1% TritonX-100 and obtain precipitated proteins. The precipitate was suspended in30 ml of buffer 1 with stirring and recentrifuged to obtain insolubleproteins.

UB-E1 protein having a purity of at least 60% was obtained by the abovesimple washing procedure.

Step 6: Dissolution and Fractionation of Insoluble Precipitate

The insoluble precipitate comprising UB-E1 proteins obtained in Step 5was suspended in 50 ml of buffer 2 containing 8M guanidine HCl (50 mMTris, pH 9.0, 1 mM EDTA, 2 mM β-mercaptoethanol). The suspension wasstirred at a room temperature for 30 minutes and centrifuged at 11,000rpm for 25 minutes with centrifuge to remove the insoluble precipitateand obtain the supernatant. The supernatant was diluted with buffer 2 tohave the final concentration of 0.5M guanidine HCl; and centrifuged toremove the supernatant and obtain a precipitate containing the UB-E1protein.

Step 7: Dissolution of Insoluble Precipitate

The insoluble precipitate comprising UB-E1 protein obtained in Step 6was suspended in 20 ml of buffer 3 (50 ml sodium carbonate, pH 9.5, 1 mMEDTA, 2 mM β-mercaptoethanol) containing 8M urea. The suspension wasstirred at a room temperature for 1 hour to remove the insolubleprecipitate and obtain the supernatant by centrifugation at 11,000 rpmfor 25 minutes (Beackman J2-21, Rotor JA14).

Step 8: Q-Sepharose Ion Exchange Chromatography

The supernatant obtained in Step 7 was passed over Q-Sepharose column(Pharmacia, FF, 1.2 cm×7 cm) equilibrated with said buffer 3; and 100 mlof the buffer having a concentration gradient of 0 to 0.4M sodiumchloride was added to elute the bound proteins. The protein fraction wassubjected to electrophoresis on 15% SDS-polyacrylamide gel to collect afraction comprising UB-E1 protein and obtain UB-E1 protein having apurity of at least 90%.

(6-D): Purification of KHCV UB-CORE 14 Protein

Step 1: Culture of Recombinant E. coli

E. coli W3110 ptrpH-UB-CORE 14(ATCC 68642) transformed with a vector(ptrpH-UB-CORE 14) containing cDNA fragment of KHCV and ubiquitin genewas cultured in LB medium containing 50 μg/ml ampicillin and 100 μg/mltryptophan at 37° C. for 12 hours; 50 n of the culture was transferredto 1 l M9 medium and cultured at 37° C. for 6 to 8 hours; and collectedcell paste in accordance with the same process as in Step 1 of Example(6-A).

Step 2: Disruption of Cells

4 g of E. coli cells obtained in Step 1 was suspended in 20 ml of abuffer (50 mM Tris, pH 7.5, 5 mM EDTA, 10 mM β-mercaptoethanol, 1 mMphenylmethylsulfonylfluoride, 1 μg/ml pepstatin) at 4° C. 4 mg oflysozyme was added to the suspension, stirred for 5 minutes andsubjected to ultrasonic treatment in ice-bath for 20 minutes with anultrasonicator to disrupt the cells.

Step 3: Identifiction of Specific Antigen Protein

A small amount of the homogenate obtained in Step 2 was subjected toelectrophoresis on 15% SDS-polyacrylamide gel as described in theprevious section and stained with Coomassie brilliant blue. The resultwas indicated that the proteins about 23,000 dalton (hereinafterreferred to as KHCV UB-CORE 14 protein) was expressed.

Subsequently, the proteins separated in the above SDS-PAGE were blottedonto a nitrocellulose filter. The filter was subjected towestern-blotting in accordance with the same process as in Step 3 ofExample (5-A). The result showed that the KHCV UB-CORE 14 protein in theentire E. coli homogenate was immunologically reacted with the serum ofa hepatitis C patient to exhibit a visible band.

Step 4: Treatment with Urea

The homogenate obtained in Step 2 was centrifuged at 12,000 rpm for 20minutes with a centrifuge (Beckman J2-21, Rotor JA 20) to remove theinsoluble material and obtain the supernatant. 9M urea was added to thesupernatant to the final concentration of 8M and stirred for 12 hours atroom temperature.

Step 5: Treatment with Acid

To the solution obtained in Step 4 was added 1M sodium acetate (pH 4.5)to be the final concentration of 10 mM, followed by an addition of 1Macetic acid to be pH=5.0 with stirring for 1 hour at room temperature.The solution was centrifuged at 11,000 rpm with a centrifuge (BeckmanJ2-21, Rotor JA 14) to remove the precipitate and obtain thesupernatant.

Step 6: CM-ion Exchange Chromatography

The solution containing KHCV UB-CORE 14 protein obtained in Step 5 waspassed at a flow rate of 1 ml/min. over a column (2.5 cm×10 cm) having25 ml of CM-Sepharose resin (Pharmacia, Sweden) equilibrated with abuffer (pH 5.0) containing 8M urea, 1 mM EDTA, 10 mM β-mercaptoethanoland 10 mM acetate. Materials remaining in the column in free form werethoroughly washed with said equilibrating buffer solution. Proteinsbound to in the column were eluted at a flow rate of 3 ml/min. with 500ml of said equilibrating buffer solution having a concentration gradientof 0 to 0.5M sodium chloride. The eluate was subjected to SDS-polyacrylamide gel electrophoresis, which indicated that KHCV UB-CORE 14 proteinwas eluted at about 0.3M. The fractions containing KHCV UB-CORE 14 werecollected for use in the next step.

Step 7: S-200 Gel Permeation Chromatography

The fractions collected in Step 6 were passed over YM5 ultrafiltrationmembrane (Amicon, U.S.A.) to concentrate to a volume of 10 nm. Theconcentrate was passed over S-200 Sephacryl column (2.5 cm×100 cm,Pharmacia, Sweden) equilibrated with PBS solution containing 6M urea, 1mM EDTA and 1 mM β-mercaptoethanol at a flow rate of 0.5 ml/min toseparate proteins according to their molecular weight. The proteinfractions were subjected to SDS-polyacrylamide gel electrophoresis.Fractions comprising KHCV UB-CORE 14 protein were collected.

Step 8: Mono-S Chromatography

The solution of KHCV UB-CORE 14 protein obtained in Step 7 was furtherpurified by passing it over FPLC Mono-S column (HR 5/5, Pharmacia,Sweden). The KHCV UB-CORE 14 protein solution was diluted with the samevolums of buffer A, passed over the column equilibrated with buffer A(pH 7) containing 6M urea, 1 mM EDTA, 1 mM β-mercaptoethanol and 10 mMphosphate, which was then washed with said buffer A. Thereafter, bufferB containing 6M urea, 1 mM EDTA, 1 mM β-mercaptoethanol, 10 mM phosphateand 0.5M sodium chloride was added gradually to an amount of 35% forfirst 5 minutes, 70% for next 55 minutes and 100% for final 10 minutesat a flow rate of 0.8 ml/min to elute the bound proteins. The KHCVUB-CORE 14 protein was eluted when the amount of buffer B reached 60%,i.e., when the concentration of sodium chloride became 0.25M.

The fraction was dialyzed against PBS solution at 4° C. to obtain 4 mgof KHCV UB-CORE 14 protein having a purity of at least 90%.

(6-E): Purification of UB-E2N Protein

Step 1: Culture of Recombinant Bacterial Cell

E. coli W3110 ptrpH-UB-E2N(ATCC 68966) which is capable of producing afused protein of KHCV E2N protein and ubiquitin was cultured withshaking for 12 hours in LB medium containing 50 μg/ml ampicillin. 10 mlof the culture was transferred to 1 l of M9 medium containing 2%casamino acid and 10 μg/ml of tryptopan; and cultured with shaking at37° C. for about 3 hours. To the culture was added indoleacrylic acid(IAA) to be the final concentration of 50 μg/ml when its O.D. at 650 nmwas 0.2 to induce the production of recombinant UB-E2N protein. Afterabout 5 hours from the addition of IAA, the culture was centrifuged at3,500 rpm for 25 minutes with a centrifuge (Beckman J-6B, Rotor JS4.2)to collect the cell precipitate. The precipitate was washed once withPBS.

Step 2: Identification of the Specific Antigen

The homogenate was subjected to electrophoresis on 15%SDS-polyacrylamide gel. The result indicated that UB-E2N protein wasexpressed in a molecular weight of about 28,000 dalton.

Subsequently, proteins separated on the gel were blotted onto aImmobilone P filter (Millipore, Cat. No. IPUH 00010, pore size 0.45 μm).The filter was placed into PBS (10 mM phosphate, pH 7.0, 0.15M sodiumchloride) containing 0.5% Tween 20 and shaken at a room temperature for2 hours to block a non-specific binding of immunoglobulin G. 10 ml ofthe serum of a hepatitis C patient as described previously diluted withPBS containing 0.5% gelatin and 0.05% Tween in a ratio of 1:20 was addedthereto. The resultant was reacted with mild shaking at a roomtemperature for 1 hour and washed with four times for 5 minutes eachwith PBS containing 0.05% Tween 20. Anti-human immunoglobulin G labelledwith an alkaline phosphatase (Boehringer Manheim, Cat. No. 605 415,Anti-Human IgG-ALP) was diluted with PBS containing 0.5% gelatin and0.05% Tween 20 in a ratio of 1:1000 and 10 ml of the diluted solutionwas added to the filter. The resultant was reacted with shaking at roomtemperature for 1 hour and washed four times with PBS containing 0.05%Tween 20 and two times with 100 mM Tris buffer (pH 9.5, 5 mM magnesiumchloride, 100 mM sodium chloride) for 5 minutes each.

To the filter was added 100 mM Tris buffer containing 125 μg/ml of nitroblue tetrazorium (Pierce, NBT) and 25 μg/ml of bromo chloro indolephosphate (Pierce, BCIP) to develop a color reaction. As a result, theUB-E2N protein in the entire cell homogenate was immunologically reactedwith the serum of a hepatitis C patient to produce a visible band.

Step 3: Disruption of Cells and Removal of Soluble Protein

About 3 g of the cell precipitate obtained in Step 1 was suspended in 50ml of buffer 1 (20 CM Tris, pH 7.5, 1 mM EDTA, 2 mM β-mercaptoethanol, 1mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A); and lysozymesolution was added to the final concentration of 0.2 mg/ml, reacted at37° C. for 30 minutes and subjected to ultrasonic treatment in ice at anoutput of 70% for 5 minutes with an ultrasonicator to disrupt the cellsand obtain the lysate. The homogenate was centrifuged at 11,000 rpm for25 minutes with a centrifuge (Beckman J2-21, Rotor JA 14) to removesoluble proteins and obtain the insoluble precipitate.

Step 4: Washing of Insoluble Precipitate with Triton X-100 and TrisBuffer

The precipitate obtained in Step 3 was suspended in 30 ml of buffer 1(20 mM Tris, pH 7.5, 1 mM EDTA, 2 mM β-mercaptoethanol) containing 1%Triton X-100. The suspension was stirred at a room temperature for 30minutes and centrifuged at 11,000 rpm for 25 minutes with a centrifuge(Backman J2-21, Rotor JA14) to remove a soluble proteins and obtain theprecipitated protein. The precipitate was suspended in 30 ml ofbuffer 1. The suspension was stirred and recentrifuged to obtaininsoluble proteins.

The UB-E2N protein having a purity of at least 70% was obtained throughthe above simple washing procedure.

Step 5: Dissolution of Insoluble Precipitate with 8M Urea

The insoluble precipitate comprising UB-E2N protein obtained in Step 4was suspended in 40 ml of buffer 2 (50 mM Tris, pH 9.0, 1 mM EDTA, 2 mMβ-mercaptoethanol) containing 8M urea. The suspension was stirred atroom temperature for 1 hour and centrifuged to remove the insolubleprecipitate and obtain the supernatant.

Step 6: S-200 Gel Permeation Chromatography

40 ml of 8M urea solution comprising UB-E2N obtained in Step 5 wasconcentrated to a volume of 5 ml with YM10 ultrafiltration membrane(Amicon), passed at a flow rate of 40 ml/hour over S-200 resin column(2.5 cm×90 cm, Pharmacia, U.S.A.) equilibrated with buffer 2 containing4M urea, and collected fractions with 2 ml/tube. The fractions weresubjected to electrophoresis on SDS polyacrylamide gel to pool thefractions comprising UB-E2N protein.

Step 7: Q-Sepharose Ion Exchange Chromatography

The solution comprising UB-E2N protein obtained in Step 6 was passedover Q-Sepharose column (FF, 1.2 cm×7 cm, Pharmacia, U.S.A.)equilibrated with buffer 2 containing 4M urea; and 150 ml of the bufferhaving a concentration gradient of 0 to 1.0M sodium chloride was addedto elute bound proteins. The fractions were subjected to electrophoresison SDS-polyacrylamide gel to collect fractions of the UB-E2N having apurity of at least 80%.

Step 8: Removal of Urea and FPLC-phenyl Chromatography

4M urea solution comprising UB-E2N protein obtained in Step 7 wasconcentrated to a volume of 8 ml with YM 10 ultrafiltration membrane(Amicon) and dialyzed against buffer 3 (20 mM Tris, pH 9.0, 1 mM EDTA, 2mM β-mercaptoethanol, 0.2M sodium chloride) using a dialysis membrane(Spectrum Medical Industries, Inc., M.W. cut off 6,000-8,000) to removethe urea. To the solution was added sodium chloride to a finalconcentration of 1M. The resultant was passed over FPLC-phenyl Sepharosecolumn (Pharmacia, HR 5/5, 0.5 cm×5 cm); and 40 ml of the buffer havinga concentration gradient of 1.0M to 0 M sodium chloride was added toelute bound proteins. The fractions were subjected to electrophoresis onSDS-polyacrylamide gel to pool the fractions comprising UB-E2N proteinhaving a purity of at least 90%.

(6-F): Purification of UB-E2C Protein

Step 1: Culture of Recombinant Cells

E. coli W3110 which is capable of producing a fused protein of KHCV E2Cprotein and ubiquitin was cultured with shaking for 12 hours in LBcontaining 50 μg/ml of ampicillin. 20 ml of the culture was transferredto 1 l of M9 medium containing 2% casamino acid and 10 μg/ml oftryptopan cultured with shaking at a temperature of 37° C. for about 2hours. To the culture was added indoleacrylic acid (IAA) to a finalconcentration of 50 μg/ml when the O.D. at 650 nm was 0.3 to induce theproduction of recombinant UB-E2C protein. After about 3 hours from theaddition of IAA, the culture was centrifuged at 3,500 rpm for 25 minuteswith a centrifuge (Beckman J6, Rotor HS4) to collect the cellprecipitate. The precipitate was washed once with PBS.

Step 2: Identification of Specific Antigen

The precipitate was subjected to electrephoresis on 15% SDSpolyacrylamide gel. The result indicated that the UB-E2C protein wasexpressed in a molecular weight of about 25,000 dalton.

Subsequently, proteins separated on gel were blotted onto Immobilone PFilter (MILLIPORE, cat. #. IPUH 00010, pore size 0.45 μm). The filterwas placed into PBS containing 0.5% Tween 20 and shaken at roomtemperature for 2 hours to block a non-speific binding of immunolobulinG. 10 ml of the serum from a hepatitis C patient diluted with PBScontaining 0.5% gelatin and 0.05% Tween in a ratio of 1:20 was addedthereto. The resultant was mildly shaken at room temperature for 1 hourand washed with four times for 5 minutes each with PBS containing 0.05%Tween 20. Anti-human immunoglobulin G labelled with horseradishperoxidase (Bio-Rad Lab. Anti-Human IgG-HRP) was diluted with PBScontaining 0.5% gelatin and 0.05% Tween 20 in a ratio of 1:500 and 10 mlof the diluted solution was added to the filter. The resultant wasreacted with shaking at room temperature for 1 hour and washed fourtimes with PBS containing 0.05% Tween 20 and two times with 50 mM Trisbuffer (pH 7.0) for 5 minutes each.

To the filter was added 50 mM Tris buffer containing 400 μg/ml4-chloro-1-naphtol and 0.03% hydrogen peroxide to develop colorreaction. As a result, the UB-E2C protein in the entire cell homogenatewas immunologically reacted with the serum of a hepatitis C patient toexhibit a visible band.

Step 3: Disruption of Cells and Removal of Soluble Protein

About 1 g of the cell precipitate obtained in Step 1 was suspended in 50ml of a lysis buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 2 mMβ-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and 1 μg/mlpepstatin A); and lysozyme solution was added to a final concentrationof 0.5 mg/ml, incubated at 37° C. for 30 minutes and subjected toultrasonic treatment in ice at an output of 70% for about 5 minutes withan ultrasonicator to disrupt cells and obtain a homogenate. Thehomogenate was centrifuged at 11,000 rpm for 25 minutes with acentrifuge (Beckman J2-21, Rotor JA 14) to remove soluble proteins andobtain an insoluble precipitate.

Step 4: Washing of Insoluble Precipitate with Triton X-100 and TrisBuffer

The precipitate obtained in Step 3 was suspended in

20 ml of buffer 1 (20 mM Tris, pH 7.5, 1 mM EDTA, 2 mMβ-mercaptoethanol) containing 1% Triton X-100. The suspension wasstirred at room temperature for 30 minutes and centrifuged at 11,000 rpmfor 25 minutes with a centrifuge (Beckman J2-21, Rotor JA14) to removesoluble proteins and obtain precipitated proteins. The precipitate wassuspended in 30 ml of buffer 1. The suspension was stirred andrecentrifuged to obtain insoluble proteins.

Step 5: Dissolution of Insoluble Precipitate with 8M Urea

The insoluble precipitate comprising UB-E2C protein obtained in Step 4was suspended in 20 ml of buffer 2 (50 mM carbonate, pH 9.5, 1 mM EDTA,2 mM β-mercaptoethanol) containing 8M urea. The suspension was stirredat a room temperature for 1 hour and centrifuged to remove the insolubleprecipitate and obtain the supernatant.

Step 6: FPLC-Mono Q Ion Exchange Chromatography

The supernatant obtained in Step 5 was passed over FPLC-Mono Q column(Pharmacia, HR 5/5, 0.5 cm×5 cm, U.S.A.) equilibrated with buffer 2containing 0.1M sodium chloride; and then 40 ml, of the buffer having aconcentration gradient of 0.1 to 0.4M sodium chloride was added to elutebound proteins. The fractions were subjected to electrophoresis onSDS-polyacrylamide gel to pool the fractions having a purity of at least80%.

Step 7: Removal of Urea and FPLC-phenyl Chromatography

8M urea solution comprising UB-E2C protein obtained in Step 6 wasconcentrated to a volume of 14 ml with YM 10 ultrafiltration membraneand dialyzed against buffer 3 (20 mM Tris, pH 9.0, 1 mM EDTA, 2 mMβ-mercaptoethanol, 0.2M s odium chloride) using a dialysis membrane(Spectrum Medical Industries, Inc., M.W. cut off 6,000-8,000) to removethe urea. To the solution was added sodium chloride to a finalconcentration of 1M. The resultant was passed over FPLC-phenyl Sepharosecolumn (Pharmacia, HR 5/5, 0.5 cm×5 cm); and 40 ml of the buffer havinga concentration gradient of 1M to 0 M sodium chloride was added to elutethe bound proteins. The fractions were subjected to electrophoresis onSDS-polyacrylamide gel to pool the fractions comprising UB-E2C proteinhaving a purity of at least 90%.

EXAMPLE 7 Detection of Anti-KHCV Antibodies to KHCV Recombinant Proteins

(7-A): Reactivity of Mixed Positive and Negative Serum Sample vs.Concentration of Antigen

Each of KHCV 403, KHCV 897 and KHCV UB-CORE 14 protein was dilutedserially in two folds with 50 mM sodium borate buffer (pH 9.0) from aconcentration of 0.25 μg/ml, 2.0 μg/ml and 2.0 μg/ml, respectively. Thediluted protein solutions were added to the wells of a microtiter plate(Dynatech, Immulon type 1 microtiter plate) in an amount of 200 μl/welland incubated at 37° C. for 2 hours wherein the plate was covered with apara-film to minimize evaporation of the solution.

The plate coated for 2 hours was washed once with PBS containing 0.05%(v/v) Tween-20 (pH 7.4, hereinafter referred to as the washingsolution). PBS containing 0.1% gelatin (v/v) was added to the wells inan amount of 210 μl/well; and was incubated at 37° C. for 2 hours. Thewells were washed twice with 300 μl of said washing solution; and 190 μlof PBS containing 0.25% gelatin, 1 mM EDTA, 1.0% (v/v) Triton X-100 and0.02% Thimerosal; and 10 μl of a positive serum sample of a HCV patientor a negative serum sample was added to every well and mixed for severalseconds; and incubated at 37° C. for 1 hour. The positive serum sampleof a HCV patient and the negative sample used were tested by adiagnostic kit for hepatitis C using C-100 antigen which is manufacturedby Ortho Diagnostic Systems, Raritan, N.J., 88869, U.S.A, respectively,prior to use. The serum samples were supplied by Severance Hospitalattached to Yonsei University located in Korea.

The wells which were reacted at 37° C. for 1 hour were washed five timeswith 300 μl of the washing solution; and anti-human IgG γ-chainimmunoglobulin labelled with horseradish peroxidase (HRP) (Bio-RadCompany, Richmond, Calif. 94804, U.S.A, 0.1 mg protein/ml) was dilutedin 5000 folds with PBS containing 10% fetal bovine serum, 1% Ficoll(Sigma, v/v), 0.02% Thimerosal and 0.05% Tween-20; and the dilutedsolution was added to the wells in an amount of 200 μl/well. Theresultant was incubated at 37° C. for 1 hour and washed 5 times withsaid washing solution. Thereafter, 200 μl of O-phenylene diaminedihychloric acid (OPD, Sigma, 10 mg/ml) which was dissolved in 50 mMcitrate buffer and was adjusted to pH 5.5 by adding phosphate was addedto each well and incubated at room temperature for 30 minutes in thedark. To the resultant was added 50 μl of 4N sulfuric acid per each wellto stop the color development; and O.D. of each well was determined atthe wavelength of 492 nm with Dynatech Microtiter Plate Reader (see FIG.19).

(7-B): Preparation of Diagnostic Kit

The antigens of purified KHCV UB-CORE 14, KHCV 897 and KHCV 403 proteinwere used to prepare a diagnostic kit. The antigens may be diluted to anoptimum concentration with 10 mM sodium carbonate buffer (pH 9.5) or 50mM sodium borate buffer (pH 9.0); added to the wells of Immulon type 1microtiter plate comprising 96 wells (Dynatech) in an amount of 150 to200 μl/well; and incubated at a temperature of 4° C. for 12 to 18 hoursto allow the antigen to adsorb to the walls of plate.

The optimum concentrations of each antigens are 0.18 to 0.75 μg/ml forKHCV UB-CORE 14 protein, 0.06 to 0.3 μg/ml for KHCV 897 protein and 0.12to 0.5 μg/ml for KHCV 403 protein. 0.3 μg/ml of each antigen was used inthis example.

The content of each after coating well was removed with an aspirator.The plate was washed with PBS (PBS, pH 7.4) containing 0.05% (v/v)Tween-20 and blocked with PBS (210 μg/well) (pH 7.4) containing 0.1%(w/v) gelatin for 2 hours at 37° C. and washed with said washingsolution 3 times. The moisture remained in the wells was removed with anabsorption apparatus.

190 μl of a buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Triton X-100,0.1 mM EDTA, 0.02% Thimerosal) containing 1% (v/v) bovine serum and 10μl of sample to be tested were added to each well and incubated at 37°C. for 1 hour to induce a binding reaction of HCV antibody in a samplewith antigen adsorbed in the wells. The plate was washed five times withPBS (pH 7.4) containing 0.05% (v/v) Tween 20; and 200 μl of ananti-human IgG-HRP (Goat anti-human IgG-HRP, Bio-Rad Lab., U.S.A.) whichwas diluted with a buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.02%Thimerosal, 1% Ficoll) containing 10% (v/v) bovine serum albumin wasadded thereto and incubated at 37° C. for 1 hour followed by washingwith PBS (pH7.4) containing 0.05% (v/v) Tween 20. 200 μl of OPD solutionwas added to develop a color reaction at room temperature for 30minutes. Thereafter, 50 μl of 4N sulfuric acid per well was added tostop the reaction and then the O.D. was determined at a wavelength of492 nm. The cut-off value which is a standard value for determination ofpositivity or negativity was settled as 0.4 plus average absorbance(O.D.) of the negative sample.

The results for each KHCV protein and mixed antigen in accordance withthe above are represented in Table 1. The comparative HCV diagnosticreagent was commercially available from Ortho Diagnostic Systems andused in accordance with the manufacturer's instruction.

TABLE 1 Reactivity of KHCV proteins to the antibodies against KHCVdetermined by Enzyme Immunoassay Antigen Mixed Ortho Antigen KHCVAntigen Antigen HCV Sample KHCV 897 UB-CORE KHCV 403 (of threeDiagnostic No. protein 14 protein protein proteins) Kit 1 ++ +++ − ++++− 2 ++++ ++++ ++ ++++ + 3 + − − ++ − 4 + + − ++ − 5 ++++ ++++ ++++++++ + 6 ++ − − +++ − 7 ++++ +++ − ++++ + 8 − ++ − +++ − 9 − − +++ ++++− 10  − +++ − ++++ − 11  ++ + − +++ − 12  ++++ +++ +++ ++++ + 13  ++ − −++ − Note: 1) ++++: Cut off value + 1.5 ≦ absorbance(O.D.) +++: Cut offvalue + 1.0 ≦ absorbance < Cut off value + 1.5 ++: Cut off value + 0.5 ≦absorbance < Cut off value + 1.0 +: Cut off value ≦ absorbance < Cut offvalue + 0.5 −: absorbance < Cut off value 2) Cut off value was 0.32 forKHCV 897 protein, 0.27 for KHCV UB-CORE 14 protein, 0.35 for KHCV 403protein, 0.483 for mixed antigens and 0.453 for Ortho diagnostic kit,respectively. 3) Ortho HCV diagnostic kit was commerically availablefrom Ortho Diagnostic Systems, U.S.A.

(7-C): Accuracy of Diagnosis

To demonstrate the accuracy of the result of the present diagnosis, 17serum samples which had been diagnosed as positive by using thediagnostic kit for hepatitis C manufactured and sold by Ortho DiagnosticSystems were diagnosed again with the diagnostic kit of the presentinvention; and also with the immunoblotting kit (Chiron RIBA HCV TestSystem, 2 nd Generation, manufactured by Ortho Diagnostic Systems,U.S.A., Product Code 933491) which is recommended as a confirmationassay and comprises 4 antigens except one SOD control antigen (see Vander Poel, C. L. et al., Lancet, 337, 317-319 (1991)). These results aresummarized in Table 2, which show that the diagnostic method of thepresent invention has a lower false positive than Ortho's diagnostic kitfor hepatitis C.

TABLE 2 Comparison of Diagnosis with Ortho's 2nd GenerationImmunoblotting Kit and the Present Diagnostic Kit Present Antigens ofOrtho 2nd Generation Diagnostic Sample Immunoblotting Kit Judg- Kit**No. 5-1-1 C100-3 C33c C22-3 SOD ment* (7-B)  1** +/− +/− − − − − − 2++++ ++++ ++++ ++++ − + ++++ 3 + +/− ++++ ++++ − + ++++ 4 + ++++ +++++/− − + ++++ 5 − − − − − − − 6 − − − − − − − 7 − +/− − − − − − 8 − − − −− − − 9 − +/− − − − − − 10  − − − − − − − Positive ++ ++++ ++++ ++ − +++++ Control Negative − − − − − − − Control *If a sample found to havemore than one +, i.e. , show a positive reaction in at least twoantigens except the SOD control antigen, then it was judged to bepositive. **Mixed antigen obtained from Example(7-B) was used as thereagent.

EXAMPLE 8 Determination of Presence of Hepatitis C Virus with PolymeraseChain Reaction Using Probe

(8-A): Extraction of RNA of Hepatitis C Virus

To 100 μl of a serum to be tested were added 100 μl of TNE solution (100mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.2M NaCl), 300 μl of RNAzol solution(TM Cinna Scientific, Inc., Tex. 77546, U.S.A) and 300 μl of chloroformwhich was mixed with shaking thoroughly. The resultant was centrifugedat 15,000 rpm and at a temperature of 4° C. for 5 minutes with Eppendorfmicrofuge to form a precipitate. The supernatant was collected andextracted with 300 μl of phenol and 300 μl of chloroform. The extractwas precipitated and the precipitate was dissolved in 10 μl of TE buffer(10 mM Tris HCl, pH 8.0, 0.1 mM EDTA) and stored at a temperature of−70° C.

(8-B): Determination of Presence of Hepatitis C Virus with PolymeraseChain Reaction

RNA extracted in the above was mixed with 4 μl of distilled water and 1μl of 0.1M CH₃HgOH and left at a room temperature for 10 minutes. 0.5 μlof 1M β-mercaptoethanol, 10 μl of RNasin, 5 μl of 5×RT buffer (BRL,Gaithersburg, Md., 20877, U.S.A), 1.25 μl of dNTP (10 mM dGTP, dTTP,dCTP and dATP), 1 μg of random primer, 1.25 μl (18 unit/μl) ofSuperscript H⁻Reverse Transcriptase (BRL, U.S.A.) were added thereto;and then, distilled water was added to a total volume of 25 μl andreacted at a temperature of 42° C. for 1 hour. After the reaction, theresultant was heated at a temperature of 65° C. for 15 minutes toinactivate enzymes and used for polymerase chain reaction.

A first polymerase chain reaction was carried out as follows. 0.5 μl ofAmplitaq DNA polymerase (Perkin Elmer Cetus, U.S.A.) was mixed with 10μl of 10×Taq polymerase buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 155mM MgCl₂, 0.1% (w/v) gelatin), 10 μl of a mixture of 1.25 mM dNTPs, 2 μgof primer A (SEQ ID NO: 125)of 5′-CATAGTGGTCTGCGGAACCG-3′, 2 μg ofprimer B (SEQ ID NO: 126) of 5′-TTGAGGTTTAGGATTCGTGC-3′ and 75 μl ofdistilled water. 50 μl of mineral oil was added thereto to preventevaporation of the solution; and, the first PCR was carried out byrepeating 40 times the thermal cycle of: 95° C. for 2 minutes, 55° C.for 2 minutes and 72° C. for 3 minutes.

A second PCR was carried out by repeating twenty times under the samecondition as the first PCR after mixing 1 μl of the product of the firstPCR with 1 μl of primer C (SEQ ID NO: 127) of 5′-TACACCGGAATTGCCAGGAC-3′and 1 μl of primer D (SEQ ID NO: 128) of 5′-TCATGGTGCACGGTCTACGAG-3′.

About 5 μl the second of PCR product was subjected to 7% polyacrylamidegel electrophoresis to determine the presence of hepatitis C viruswherein the positive sample exhibited a DNA band of 182 bp.

EXAMPLE 9 Preparation of Specific Antibody Against Hepatitis C Antigenof KHCV Protein

(9-A): Immunization

KHCV 897 protein dissolved in saline was mixed with an equivalent amountof Freund's complete adjuvant; and 0.2 ml of a mixture containing 50 μgof the protein was injected intraperitoneally to about 10 week oldBalb/c mouse. 30 μg of the protein mixed with Freund's incompleteadjuvant was injected at intervals of 2 to 3 weeks. After 2 weeks of thesecond injection, small amount of blood was drawn from the tail of themouse and subjected to an enzyme immunoassay to determine the antibodytiter. 50 to 100 μg of the protein 0.5 ml of saline was further injectedwhen the titer reached to 10,000. Antibody titer was operationallydefined as that dilution of serum that resulted in 0.2 absorbance unitsbackground in ELISA procedure. After 3 to 4 days, spleen cells of themouse were used for the preparation of a cell producing monoclonalantibody.

(9-B): Cell Fusion

Immunized spleen cells were fused with P3×63-Ag8.653(ATCC CRL 1580)which was a myeloma cell of mouse. 5×10⁷ spleen cells of immunized mousewas mixed with 2×10⁷ P3×63-Ag8.653 and centrifuged at 300×g for 10minutes. The cell precipitate was washed with IMDM medium (Gibco,U.S.A.) and centrifuged. The supernatant was discarded and 1 ml of 50%PEG (Kodak, molecular weight of 1450 dalton) solution was added dropwiseover one minute to the cell precipitate with stirring. The resultant wascentrifuged at 200×g for two minutes; and 5 ml of IMDM medium was addedslowly over three minutes, followed by the addition of 5 ml of IMDMmedium containing 10% fetal bovine serum over five minutes withstirring.

IMDM medium containing 10% fetal bovine serum was added thereto to atotal volume of 50 ml and centrifuged for 10 minutes.

The supernatant was discarded; and IMDM-HAT medium prepared by adding10% fetal bovine serum, 100 μM hypoxanthine, 0.4 μM amino-pterin and 16μM thymidine to IMDM medium was added thereto to dilute the cellconcentration to be 5×10 ⁵ cells of P3×63-Ag 8.653 per ml. The resultantwas added to a plate (96 wells) for tissue culture in an amount of 0.1ml/well. 0.1 ml IMDM-HAT medium containing 1×10⁵ cells/ml ofintraperitoneal macrophage was added to the wells and cultured 1 dayprior to the fusion. The myeloma cell and unfused spleen cell cannotgrow in HAT medium.

Accordingly, the cells grown in the medium were considered to be thefused cells. An assay of antibody activity was carried out with thesupernatant which was sampled when the hybridoma was grown to a level of10 to 50%.

(9-C): Screening of Titer of Monoclonal Antibody

Titration of monoclonal antibodies produced in Step (9-B) was carriedout in accordance with the following enzyme immunoassay.

Step 1

KHCV 897 protein was dissolved in 50 mM sodium borate buffer (pH 9.0) toa concentration of 2 μl/ml. 100 μl of the solution was added to eachwell of Immulon type I plate (Dynatech) and incubated at a temperatureof 37° C. for 2 hours.

Step 2

The wells were washed once with PBS (pH 7.4) containing 0.05% Tween-20(v/v) (hereinafter referred to as the washing solution); and 200 μl ofPBS containing 0.1% gelatin (w/v) was added thereto to block theadsorption sites of the proteins which remained in the well at atemperature of 37° C. for 1 hour.

Step 3

The wells of Step 2 were washed twice with the washing solution; and 50μl of PBS containing 0.25% gelatin (w/v), 1.0 mM EDTA, 1% Triton X-100(v/v) and 0.02% Thimerosal was added thereto. 50 μl of the supernatantwherein the fused cells had been grown was added to each well andincubated at a temperature of 37° C. for 1 hour.

Step 4

The wells treated in Step 3 were washed five times with the washingsolution. Anti-mouse IgG-HRP (Boehringer Manheim, Cat. No. 605-250)labelled with horseradish peroxidase (HRP) was diluted with PBScontaining 10% (v/v) fetal bovine serum, 1% (v/v) Ficoll, 0.02% (v/v)Thimerosal and 0.05% (v/v) Tween-20 in a ratio of 1:5000; and thediluted solution was added to the wells in an amount of 100 μl/well andincubated at a temperature of 37° C. for 1 hour. After the reaction, theplate was washed five times with the washing liquid.

Step 5

100 μl of 50 mM citrate/phosphate buffer (pH 5.5) containing 10 mg/5 mlof O.P.D. (Sigma Chemical Co.) was added to each well and reacted at aroom temperature in the dark for 30 minutes; and 50 μl of 2N sulfuricacid was added thereto to stop the reaction. The absorbance wasdetermined at a wavelength of 492 nm. Hybridoma which exhibited thedesired antibody activity was transferred to and grown in a 6 well plateor 24 well plate wherein, if necessary, the peritoneal macrophages ofmouse may be used as a feeder layer to provide a growth factor necessaryfor the growth of the fused cell.

(9-D): Production of Antibody

4 cell lines, i.e., Lucky 1.1, 1.2, 1.3 and 1.4 which produced thedesired monoclonal antibodies were obtained.

The antibodies of the present invention were available from either thesupernatant in which clones were cultured by the conventional method orthe ascite fluid containing the clones grown in peritoneum of a Balb/cmouse.

2.5×10⁶ fused cells were injected intraperitoneally to a Balb/c mousewhich had been pretreated with 0.5 ml of Pristane (Sigma) 7 to 14 daysbefore. After 1 to 2 weeks, seroperitoneum liquid was obtained; andantibodies were isolated therefrom in accordance with a conventionalmethod.

(9-E): Detection of Characteristics of Monoclonal Antibody

The characteristics of antibodies prepared from each clone obtained inExample (9-D) were evaluated as follows.

Step 1: Antibody's Subclass

The subclass of the mouse antibodies was determined by using theHybridoma sub-Isotyping Kit (Calbiochem, U.S.A.). The results are shownin Table 3.

Step 2: Enzyme Immunoassay

200 μl of KHCV 897 protein dissolved in 50 mM sodium borate buffer in aconcentration of 2 μl/ml was added to each well of microtiter plate(Dynatech Immunolon type 1) and incubated at a temperature of 37° C. for2 hours. The plate was washed with PBS containing 0.05% Tween-20 (v/v).The antibodies obtained from each clone were purified by a conventionalmethod, adjusted to a concentration of 1 mg/ml and diluted serially intwo folds with PBS containing 0.25% gelatin (v/v), 1.0% Triton X-100,0.02% Thimerosal and 1 mM EDTA. 210 μl of PBS containing 0.1% gelatinwas added to each well and incubated at a temperature of 37° C. for 1hour. The plate was washed with the washing solution.

200 μl of anti-mouse IgG (Boehringer Manheim, Cat. No. 605-250) labelledwith horseradish peroxidase which was dissolved in PBS containing 10%FBS (v/v), 1% Ficoll (v/v) and 0.05% (v/v) Tween-20 was added to eachwell and incubated at a temperature of 37° C. for 1 hour. Thedevelopment reaction was carried out in the same manner as in Example(9-C). The EIA efficiency of each antibody was determined as areciprocal number of the dilution fold when the O.D. value at 495 nm wasmore than 1.0. The results are given in Table 3.

Step 3: Determination of Molecular Weight

Each clone was cultured in a plate or peritoneum of a mouse. Thesupernatant or ascite fluid obtained therefrom was subjected toprotein-G Sepharose column affinity chromatography (Pharmacia) toisolate IgG which was then subjected to SDS-PAGE to determine themolecular weight of the heavy chain and the light chain in the mouseantibody obtained above. The results are represented in Table 3.

Step 4: Determination of Epitope

The variants in which a portion of KHCV 897 cDNA was differently excisedwere constructed to encode the following proteins; and the reactivity ofthe proteins to each monoclonal antibody was examined.

(1) KHCV 897 protein: A protein comprised of amino acids 1192 to 1457 ofthe amino acid sequence encoded in KHCV-LBC1

(2) KHCV 290 protein: A protein comprised of amino acids 1192 to 1289 ofthe amino acid sequence encoded in KHCV-LBC1

(3) KHCV 430 protein: A protein comprised of amino acids 1192 to 1335 ofthe amino acid sequence encoded in KHCV-LBC1

(4) KHCV 570 protein: A protein comprised of amino acids 1192 to 1382 ofthe amino acid sequence encoded in KHCV-LBC1

(5) KHCV 652 protein: A protein comprised of amino acids 1192 to 1407 ofthe amino acid sequence encoded in KHCV-LBC1

(6) KHCV 150 protein: A protein comprised of amino acids 1408 to 1457 ofthe amino acid sequence encoded in KHCV-LBC1

(7) KHCV 257 protein: A protein comprised of amino acids 1371 to 1457 ofthe amino acid sequence encoded in KHCV-LBC1

(8) KHCV 518 protein: A protein comprised of amino acids 1285 to 1457 ofthe amino acid sequence encoded in KHCV-LBC1

A sample for SDS-PAGE was prepared by adding a buffer (Laemmli, U. K.,Nature 277, 680(1970)) to E. coli cell which expressed each KHCV cDNAfragment and was boiled at a temperature of 100° C. for 5 minutes. Thereactivity of the prepared sample to antibody was examined by an immunoblotting method (Towbin, H., J. Immunol. Methods 72, 313-340(1984)). Theresults are given in Tables 3 and 4.

It can be seen from the result that antibodies obtained from Lucky 1.1have a recognition site for amino acids 1192 to 1289 of the amino acidsequence of hepatitis C; and Lucky 1.2, 1.3 and 1.4 have a recognitionsite for amino acids 1371 to 1407. Two monoclonal antibodies whoseepitopes are different from each other may be used to prepare a kit bywhich the antigens in a serum sample can be using detected by usingSandwich Enzyme Immunoassay and the like.

TABLE 3 Characteristics of Monoclonal Antibodies of the PresentInvention Binding Site Monoclonal Antibody Molecular EIA (Amino AcidAntibody Subclass Weight Efficiency Sequence) Lucky 1.1 IgG1 162,000  ×51,200 1192-1289 Lucky 1.2 IgG1 159,700 × 102,400 1371-1407 Lucky 1.3IgG1 180,800  × 51,200 1371-1407 Lucky 1.4 IgG1 177,700   × 4001371-1407

TABLE 4 Immuno Reactivity of Excised Mutant with Antibodies AntibodyAntigen Lucky 1.1 Lucky 1.2 Lucky 1.3 Lucky 1.4 KHCV 897 + + + + KHCV290 + − − − KHCV 430 + − − − KHCV 570 + − − − KHCV 652 + − − − KHCV 150− − − − KHCV 257 − + + + KHCV 518 − + + + Negative − − − − ControlRecognition 1192-1289 1371-1407 1371-1407 1371-1407 Site of Amino Acidsequence

The cell lines of Lucky 1.1 and Lucky 1.2 were deposited on Dec. 18,1991 under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC) and were assigned Accession Nos. 10949 and10950, respectively.

EXAMPLE 10 Diagnostic Agent Comprising an Antibody against KHCV Antigen

Step 1: Labelling of Onoclonal Antibody of Lucky 1.1 with HorseradishPeroxidase

As a first step, said Lucky 1.1 cell line was labelled with horseradishperoxidase by using the known periodate method (Nakane et a!., J.Histochemcytochem., 22, 1084(1974)) as follows.

0.3 ml of 0.1M sodium periodate in a 10 mM sodium phosphate buffer (ph7.0) was added to 1.2 ml of distilled water in which 5 mg of perioxidasewas dissolved; and the mixture was reacted at a room temperature for 20minutes. The resultant was dialyzed against 1 mM sodium acetate bufferfor 16 hours. 1.5 ml of peroxidase solution was mixed with 1 ml ofantibody to be labelled which had been previously prepared by dissolvingit in 20 mM sodium carbonate (pH 9.5) in a concentration of 10 mg/ml;and the mixture was reacted at a room temperature for 2 hours. Schiffbase which was unreacted was reduced off by addition of 100 μl of 4mg/ml sodium monohydride in distilled water. The resultant was subjectedto dialysis against PBS (pH 7.4) overnight, and then passed overSephacryl S 300 chromatography column to remove monoclonal antibodieswhich were not labelled.

Step 2: Adsorption of Monoclonal Antibody of Lucky 1.2 to MicrotiterPlate

200 μl of 5 μg/ml Lucky 1.2 diluted with PBS was added to each well toallow its adsorption onto the wall of the well at a 37° C. for 2 hours.

Step 3: Blocking of Non-specific Binding

The microtiter prepared in Step 2 was washed once with PBS containing0.05% Tween-20 and 0.02% Thimerosal (hereinafter referred to as thewashing solution). 200 μl of PBS containing 0.1% gelatin was added toeach well to coat the protein adsorption site over 1 hour; and the platewas washed twice with the washing solution.

Step 4: Diagnosis of Presence of Antigen

200 μl of KHCV 897 antigen which was diluted serially in two folds from200 ng/ml with PBS containing 0.25% (w/v) gelatin, 1.0% (v/v) TritonX-100, 1 mM EDTA and 0.02% Timerosal was added to each well. Forcomparison, KHCV protein was added to a normal blood sample to aconcentration of 400 ng/ml; the normal blood sample containing the KHCVantigen was diluted serially in two folds; and 100 μl of the dilutedblood was mixed with 100 μl of said buffer and added to each well. Thiswas intended to show that the presence of an antigen of hepatitis C inblood can be detected by Sandwich Enzyme Immunoassay by using theantibodies obtained. The normal blood wherein KHCV 897 antigen was notadded was used as a negative control. The plate was incubated at atemperature of 37° C. for 1 hour and washed five times with the washingsolution.

Step 5: Screening of Antigen with Lucky 1.1 Labelled with Peroxidase

200 μl of Lucky 1.1 which was diluted to a concentration of 5 μg/ml withPBS containing 10% (v/v) fetal bovine serum, 1% Ficoll, 0.05% Tween-20and 0.02% Thimerosal was added to each well, which was incubated at atemperature of 37° C. for 1 hour.

Step 6: Color Development Reaction

The plate treated in Step 5 was washed five times with the washingsolution; and 200 μl of O.P.D. developing reagent which was prepared byadding O-phenylenediamine (Sigma) to 50 mM citrate/phosphate buffer (pH5.5) to a concentration of 2 mg/ml was added to each well and left at aroom temperature in the dark for 30 minutes to develop a color reaction.50 μl of 4N sulfuric acid was added to stop the reaction. Absorbancethereof was determined at a wavelength of 492 nm. The results arepresented in FIG. 43.

EXAMPLE 11 Screening of Antigen in Serum of a Hepatitis C Patient withSandwich Enzyme Immunoassay

100 μl of a serum to be analyzed which was mixed with 100 μl of thebuffer used in Step 4 of Example 10 was added to each well of themicrotiter prepared by the same process as in Example 10 to whichmonoclonal antibody was already adsorbed; and the antigen in the serumwas screened by the same process as in Example 10. The results are givenin Table 5.

220 samples of 231 samples (220/231) exhibited the absorbance value(O.D.) at 492 nm of less than 0.15; other 11 samples exhibited thevalues ranging from 0.15 to 0.8, which were judged to be positive. Inaccordance with Halbert's method (Halbert, S. P. et al., Clin, Chim.Acta 127, 69(1983)), the cut-off value was settled to be an absorbanceof 0.15.

Antibodies against KHCV for 15 samples including the 11 positive sampleswere screened in accordance with the same process as in Example 7. Theresults are shown in Table 6. The results may be suggest that thesandwich ELISA for KHCV 897 antigen detection is valuable and can usefor early detection of KHCV infection. Along with EIA for antibodydetection, the ELISA for antigen detection should be used for HCVpatient care and protection.

TABLE 5 Absorbance of Samples Determined by Sandwich Enzyme ImmunoassayNumber of Absortance Samples Percentage¹⁾ >= 0.5 1 0.43 0.3-0.5 3 1.300.2-0.3 4 1.73 0.15-0.2  3 1.30 <0.15 220 95.24 Total 231 100.00${\text{Note:~~}{\quad^{1)}{Percentage}\quad (\%)}}\quad = \frac{\text{The~~number~~of~~tested~~samples}}{\text{The~~number~~of~~total~~samples}}$

TABLE 6 Detection of Hepatitis C Antibody and Antigen Antibody ofAntigen of Sample Hepatitis C Hepatitis C 1 − + 2 − + 3 − − 4 − + 5 − +6 − + 7 − − 8 + − 9 − + 10  − + 11  − + 12  − + 13  − + 14  + + 15  − −Note: 1) The cut-off value was set to be an absorbance value of 0.15 forantigen diagnosis and 0.33 for antibody diagnosis.

Accordingly, KHCV proteins of the present invention, especially usingthe mixed antigen containing 3 proteins, is more reactive to theantibodies against KHCV than the commercially available HCV diagnostickit as shown in Table 1; the diagnostic kit of the present inventionproduces more accurate test results than the commercial kit; and is moreconvenient and economical than the confirmation assay kit as shown inTable 2.

While the invention has been described in connection with certainspecific embodiments, it should be recognized that various modificationsand changes as may be apparent to those skilled in the art to which theinvention pertains may be made and also fall within the scope of theinvention as defined by the claims that follow.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 128(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer RANPSHCV (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1TTTTTCATGA TTGGTGGTGG AACTGGACCG TCTCGAGNNN NNN 43 (2) INFORMATION FORSEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: oligo d(T)primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 GAGAGAGAGA GAGAGAGAGAACTAGTCTCG AGTTTTTTTT TTTTTTTTTT 50 (2) INFORMATION FOR SEQ ID NO: 3 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: Eco RI Adaptor, used as Eco RIprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 CCCCCCGAAT TCGGCACGAG 20(2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PSHCV (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4TTCATGAT TGGTGGTGGA 20 (2) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: probe oligonucleotide P652a (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 5 CATACCCG TTGAGTCTAT GGAAACTACT 30 (2)INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: probe oligonucleotide P652b (xi) SEQUENCE DESCRIPTION: SEQID NO: 6 CATTCCAA GAAGAAGTGT GACGAACTCG 30 (2) INFORMATION FOR SEQ IDNO: 7 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: probe oligonucleotideP426a (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 GAGACCTC CCGGGGCACTCGCAAGCACC 30 (2) INFORMATION FOR SEQ ID NO: 8 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: probe oligonucleotide P426b (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 8 TAATTTGG GTAAGGTCAT CGACACCCTC 30 (2)INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: probe oligonucleotide P240b (xi) SEQUENCE DESCRIPTION: SEQID NO: 9 GTCCGGGTGC TGGAGGACGG CGTGAACTA 29 (2) INFORMATION FOR SEQ IDNO: 10 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: probe oligonucleotideP513b (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 CGCATGGCCT GGGATATGATGATGAACTGG 30 (2) INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: probe oligonucleotide P810b (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 11 AAATGAGACG GACGTGCTGC TCCTTAAC 28(2) INFORMATION FOR SEQ ID NO: 12 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: probe oligonucleotide P403A (xi) SEQUENCE DESCRIPTION: SEQID NO: 12 GTGAAGAATT CGGGGGCCGG AACCTGGCAT 30 (2) INFORMATION FOR SEQ IDNO: 13 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: probe oligonucleotideP403B (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 GCTGACCTCA TTGAGGCCAACCTCTTGT 28 (2) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: probe oligonucleotide P932b (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 14 CCGGGACGTG CTTAAGGAGA TGAAGGCGAA 30(2) INFORMATION FOR SEQ ID NO: 15 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: probe oligonucleotide P496b (xi) SEQUENCE DESCRIPTION: SEQID NO: 15 CGTGTATGCG AGAAGATGGC CCTTTATGAC 30 (2) INFORMATION FOR SEQ IDNO: 16 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: probe oligonucleotideP847b (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 TGCGTGGGAG ACAGCTAGACACACTCCAG 29 (2) INFORMATION FOR SEQ ID NO: 17 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: probe oligonucleotide P798b (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 17 CTGGTTCCCG GAGCGGCATA C 21 (2)INFORMATION FOR SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: probe oligonucleotide P752a (xi) SEQUENCE DESCRIPTION: SEQID NO: 18 CCAGGTGATG ACTTTGGTCT CCAT 24 (2) INFORMATION FOR SEQ ID NO:19 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: probe oligonucleotideP675b (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 TCGATTCTTC GGTCCTGTGTGAGTGT 26 (2) INFORMATION FOR SEQ ID NO: 20 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: probe oligonucleotide P652b(2) (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 20 AAAAAGAATT CGGATCCATG ACGCGGTTGTGCGTGGTAC 39 (2) INFORMATION FOR SEQ ID NO: 21 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: probe oligonucleotide P403a(2) (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 21 CCCCCTCAGA GTCGACTCAC TTCACGTTGTCAGTGGTCAT 40 (2) INFORMATION FOR SEQ ID NO: 22 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer DA17PSHCV (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 22 TGGTGGTGGA ACTGGACCGT A 21 (2) INFORMATIONFOR SEQ ID NO: 23 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION:primer PSHCVSL (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 AAAAGTCGACTGGTGGTGGA ACTGGACCGT 30 (2) INFORMATION FOR SEQ ID NO: 24 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer KHCVR60 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 24 GTGTCCGCGC TAAGCTACTG TCC 23 (2) INFORMATIONFOR SEQ ID NO: 25 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION:primer KHCVR61 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25 TGTGGCAAGTACCTCTTCAA CTGG 24 (2) INFORMATION FOR SEQ ID NO: 26 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer KHCVL69 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 26 GTCCTGTGGG CGGCGGTTGG TGTTACG 27 (2)INFORMATION FOR SEQ ID NO: 27 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer KHCVL70 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27TTGAGGTTTA GGATTCGTGC TCAT 24 (2) INFORMATION FOR SEQ ID NO: 28 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer dC12R1RO (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 28 AGGATCCGT CGACATCGAT AATACGACTC ACTATAGGGACCCCCCCCCC CC 52 (2) INFORMATION FOR SEQ ID NO: 29 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer dT17R1RO (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 29 ATCCGT CGACATCGAT AATACGACTC ACTATAGGGATTTTTTTTTT TTTTTTT 57 (2) INFORMATION FOR SEQ ID NO: 30 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer RO (xi) SEQUENCE DESCRIPTION: SEQID NO: 30 AAGGATCCGT CGACATC 17 (2) INFORMATION FOR SEQ ID NO: 31 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer R1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 31 GACATCGATA ATACGACTCA C 21 (2) INFORMATIONFOR SEQ ID NO: 32 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION:primer NS2S1, corresponds to the strand of the fragment comprising fromthe 2776th to the 2795th nucleotides in KHCV-LBC1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 32 CGGGAGATGG CCGCATCGTG 20 (2) INFORMATION FORSEQ ID NO: 33 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primerNS2N1, corresponds to the complementary strand of the fragmentcomprising from the 3180th to the 3157th nucleotides in KHCV-LBC1 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 33 ACCTGCTAGT GCGGCCAGCT TCAT 24 (2)INFORMATION FOR SEQ ID NO: 34 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer NS2S2, includes the strand of the fragment from the2803rd to the 2822nd nucleotides in KHCV-LBC1 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 34 TTTTGGATCC GCGGTTTTTG TAGGTCTGGT 30 (2) INFORMATION FORSEQ ID NO: 35 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primerNS2N2, includes the complementary strand of the fragment from the 3159thto the 3142th nucleotides in KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 35 AAAGTCGACA TGAAGACCAT TTGGAC 26 (2) INFORMATION FOR SEQ ID NO: 36(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer NS5S1, thenucleotide sequence from the 10th nucleotide to the 3′-end correspondedto the nucleotide sequence of the fragment from the 8252nd to the 8173thnucleotides in KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36ATGGGGATCC ATATGACACC CGCTGYTTTG A 31 (2) INFORMATION FOR SEQ ID NO: 37(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer NS5N1, thenucleotide sequence from the 9th nucleotide to the 3′-end correspondedto the complementary strand of the fragment from the 8635th to the8614th nucleotides in KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37CCCCGTCGAC CTAGTCATAG CCTCCGTGAA 30 (2) INFORMATION FOR SEQ ID NO: 38(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer NS5S2, thenucleotide sequence from the 12th nucleotide to the 3′-end correspondedto the strand of the fragment from the 8278th to the 8297th nucleotidesin KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38 TTTGAGGATCCACGGTCACT GAGAAYGACA T 31 (2) INFORMATION FOR SEQ ID NO: 39 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PCOREUBI, include thestrand of the fragment from the 343rd to the 360th nucleotides inKHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39 CTTGGTGTTG AGACTCCGCGGTGGTATGAG CACGAATCCT AAACC 45 (2) INFORMATION FOR SEQ ID NO: 40 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: primer PSALCORE14, containsa stop codon to stop translation just after the 726th nucleotide ofKHCV-LBC1 (ii) MOLECULE TYPE: DNA (ix) FEATURE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 40 GGGGTCGACT ATTAGCATGT GAGGGTGTCG ATGAC 35 (2)INFORMATION FOR SEQ ID NO: 41 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: primer PSALCORE17, contains a stop codon to stop translationjust after the 852nd nucleotide of KHCV-LBC1 (ii) MOLECULE TYPE: DNA(ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41 GGGGTCGACTATTAGGGCAG ATTCCCTGTT GC 32 (2) INFORMATION FOR SEQ ID NO: 42 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: primer PSALCORE22, containsa stop codon to stop translation just after the 915th nucleotide ofKHCV-LBC1 (ii) MOLECULE TYPE: DNA (ix) FEATURE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 42 GGGGTCGACT ATTAAGCGGA ACTGGGGATG GTCAA 35 (2)INFORMATION FOR SEQ ID NO: 43 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PK403UBI, designed to initiate translation from the6649th nucleotide of KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43CTTGGTGTTG AGACTCCGGT GGTACGGGCA TGACCACTGA CAA 43 (2) INFORMATION FORSEQ ID NO: 44 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primerPK573UBI, designed to initiate translation from the 7612th nucleotide ofKHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44 CTTGGTGTTG AGACTCCGCGGTGGTACATG GACAGGCGCC CTGA 44 (2) INFORMATION FOR SEQ ID NO: 45 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: primer PK403SAL, designed tostop translation just after the 7050th nucleotide of KHCV-LBC1 (ii)MOLECULE TYPE: DNA (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45 GACTGGTCGA CTATTACTCT TGCCGCCACA AGAGGTT 37 (2) INFORMATION FOR SEQID NO: 46 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK897UBI,designed to initiate translation from the 3916th nucleotide of KHCV-LBC1(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46 CTTGGTGTTG AGACTCCGCGGTGGTGCGGT GGAATTCATA CCCG 44 (2) INFORMATION FOR SEQ ID NO: 47 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: primer PK897SAL, designed tostop translation just after the 4713th nucleotide of KHCV-LBC1 (ii)MOLECULE TYPE: DNA (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47 GACTGGTCGA CTATTAACAC GTATTACAGT CGATCAC 37 (2) INFORMATION FOR SEQID NO: 48 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: primerPK573SAL, designed to stop translation just after the 8184th nucleotideof KHCV-LBC1 (ii) MOLECULE TYPE: DNA (ix) FEATURE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 48 GACTGGTCGA CTATTAGTAC TGGAATCCGT ATGAGGAG 38(2) INFORMATION FOR SEQ ID NO: 49 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer P426B, consists of the region from the 616th to the636th nucleotides of KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49GGGTGGGCAG GATGGCTCCT G 21 (2) INFORMATION FOR SEQ ID NO: 50 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer P240B, consists of theregion from the 842nd to the 821st nucleotides of KHCV-LBC1 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 50 CCTGTTGCAT AGTTCACGCC GT 22 (2)INFORMATION FOR SEQ ID NO: 51 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer P652B, consists of the region from the 4523rd to the4560th nucleotides of KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51GTCATTCCAA GAAGAAATGT GACGAGCTCG CTGCAAAG 38 (2) INFORMATION FOR SEQ IDNO: 52 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PE2NUBI, contains25 nucleotides on the 5′-end region overlapping with the 3′-end regionof ubiquitin gene and the other nucleotides correspond to the regionfrom the 1510th to the 1530th nucleotides of KHCV-LBC1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 52 CTTGGTGTTG AGACTCCGCG GTGGTGGGGC GCAAGGTCGGGCCGCT 46 (2) INFORMATION FOR SEQ ID NO: 53 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: primer PE2NSAL, designed to stoptranslation just after the 2010th nucleotide of KHCV-LBC1 (ii) MOLECULETYPE: DNA (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53GACTGGACTA TTAATTCATC CAGGTACAAC CGAACCA 37 (2) INFORMATION FOR SEQ IDNO: 54 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PE2CUBI, contains25 nucleotides on the 5′-end region overlapping with the 3′-end regionof ubiquitin gene and the other nucleotides correspond to the regionfrom the 2011th to the 2031st nucleotides of KHCV-LBC1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 54 CTTGGTGTTG AGACTCCGCG GTGGTGGCAC TGGGTTCACCAAGACA 46 (2) INFORMATION FOR SEQ ID NO: 55 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: primer PE2CSAL, designed to stoptranslation just after the 2529th nucleotide of KHCV-LBC1 (ii) MOLECULETYPE: DNA (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55GACTGGACTA TTACGCGTCC GCCAGAAGAA GGAAGAG 37 (2) INFORMATION FOR SEQ IDNO: 56 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PEIUBI, contains25 nucleotides on the 5′-end region overlapping with ubiquitin gene andthe other nucleotides correspond to the region from the 916th to the936th nucleotides of KHCV-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56CTTGGTGTTG AGACTCCGCG GTGGTTATGA AGTGGGCAAC GCGTCC 46 (2) INFORMATIONFOR SEQ ID NO: 57 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:primer PEISAL, designed to stop translation just after the 1509thnucleotide of KHCV-LBC1 (ii) MOLECULE TYPE: DNA (ix) FEATURE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 57 GACTGGACTA TTACCCTGTC ACGTGGGTGGTGGTTCC 37 (2) INFORMATION FOR SEQ ID NO: 58 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: oligonucleotide UBI1 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 58 CCCCATATGC AAATTTTCGT CAAAACTCTA ACAGGGAAGACTATAACCCT AGAGGTTGAA 60 TCTTCCGACA CTATTGACAA CGTCAA 86 (2) INFORMATIONFOR SEQ ID NO: 59 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 91 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION:oligonucleotide UBI2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59 TAGTTGCTTACCAGCAAAAA TCAATCTCTG CTGATCCGGA GGGATACCTT CTTTATCTTT 60 GAATTTTACTTTTGACGTTG TCAATAGTCT C 91 (2) INFORMATION FOR SEQ ID NO: 60 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 98 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: oligonucleotide UBI3 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 60 ACCACCGCGG AGTCTCAACA CCAAGTGAAGAGTAGATTCC TTTTGGATGT TGTAGTCAGA 60 CAAGGTTCTA CCATCTTCTA GTTGCTTACCAGCAAAAA 98 (2) INFORMATION FOR SEQ ID NO: 61 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer PK426R (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 61 CTCCGAATTC GGTGCTTGCG AGTGCCCC 28 (2) INFORMATION FOR SEQID NO: 62 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK426X(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62 CACGCTCGAG GCATGTGAGGGTGTCGATGA C 31 (2) INFORMATION FOR SEQ ID NO: 63 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer PK513R (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 63 CTCCGAATTC GGCACGAGGC TGGAGGACGG CGTGAACT 38 (2)INFORMATION FOR SEQ ID NO: 64 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PK513X (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64CACGCTCGAG AGGCGACCAG TTCATCATCA T 31 (2) INFORMATION FOR SEQ ID NO: 65(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK810R (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 65 CTCCGAATTC GGCACGAGGG TTTCCCAGCTGTTCACCTT 39 (2) INFORMATION FOR SEQ ID NO: 66 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer PK810X (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 66 CACGCTCGAG ATTCATCCAG GTACAACCGA ACC 33 (2) INFORMATIONFOR SEQ ID NO: 67 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION:primer PK798R (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67 CTCCGAATTCGGCACGAGGG ACGTGCTGCT CCTTAAC 37 (2) INFORMATION FOR SEQ ID NO: 68 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK798X (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 68 CACGCTCGAG CAGAAGCAGC GGCCATACGC C 31 (2)INFORMATION FOR SEQ ID NO: 69 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PK754R (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69AAAAAGAATT CGGCACGAGG CTGCGAGATT GGGCTCACAC G 41 (2) INFORMATION FOR SEQID NO: 70 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK754X(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70 AAAAACTCGA GCCGCATAGTAGTTTCCATA GACTCAACGG GTATGAATT 49 (2) INFORMATION FOR SEQ ID NO: 71 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK652R (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 71 AAAAAGAATT CGGCACGAGG TTCATACCCG TTGAGTCTATGGAA 44 (2) INFORMATION FOR SEQ ID NO: 72 (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D)OTHER INFORMATION: primer PK652X (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72 ATTATTGTCG ACTATCTATC TACTCGAGTC ACAGCTTTGC AGCGAGCTCG T 51 (2)INFORMATION FOR SEQ ID NO: 73 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PK403R (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73AAAAAGAATT CACGGGCATG ACCACTGAC 29 (2) INFORMATION FOR SEQ ID NO: 74 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK403X (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 74 ATTATTCTCG AGTATCACTC TTGCCGCCAC AAGAG 35 (2)INFORMATION FOR SEQ ID NO: 75 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PK271R (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75AAAAAGAATT CACTAGCCTT ACAGGCCGG 29 (2) INFORMATION FOR SEQ ID NO: 76 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK271X (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 76 CACGCTCGAG TCACGTGACC AGGTAAAGGT C 31 (2)INFORMATION FOR SEQ ID NO: 77 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PK495R (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77CCCCCGAATT CGGCACGAGC GCTGCGGAGG AAAGCAAGTT 40 (2) INFORMATION FOR SEQID NO: 78 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer PK495X(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78 AAAAACTCGA GGACCACGTCATAAAGGGCC A 31 (2) INFORMATION FOR SEQ ID NO: 79 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer PK494R (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 79 AAAAGAATTC GGCACGAGCG ATGCATCTGG TAAAAGGGT 39 (2)INFORMATION FOR SEQ ID NO: 80 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer PK494X (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80AAAACTCGAG ATTGGAGTGA GTTTGAGCTT 30 (2) INFORMATION FOR SEQ ID NO: 81(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 105 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 81 Leu Leu Thr Leu Ser Pro His Tyr LysVal Phe Leu Ala Arg Phe 1 5 10 15 Ile Trp Trp Leu Gln Tyr Leu Ile ThrArg Thr Glu Ala His Leu 20 25 30 Gln Val Trp Val Pro Pro Leu Asn Val ArgGly Gly Arg Asp Ala 35 40 45 Val Ile Leu Leu Thr Cys Ala Val Tyr Pro GluLeu Ile Phe Asp 50 55 60 Ile Thr Lys Leu Leu Leu Ala Thr Leu Gly Pro LeuMet Val Leu 65 70 75 Gln Ala Gly Leu Ile Arg Val Pro Tyr Phe Val Arg SerGly Leu 80 85 90 Ile Arg Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly HisTyr 95 100 105 (2) INFORMATION FOR SEQ ID NO: 82 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 106 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 82 Leu Leu Thr Leu Ser Pro His Tyr Lys Val Phe Leu Ala ArgPhe 1 5 10 15 Ile Trp Trp Leu Gln Tyr Leu Ile Thr Arg Thr Glu Ala HisLeu 20 25 30 Gln Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala35 40 45 Ile Ile Leu Leu Ala Cys Ala Val His Pro Glu Leu Ile Phe Asp 5055 60 Ile Thr Lys Leu Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu 65 7075 Gln Ala Ser Ile Ile Arg Val Pro Tyr Ser Val Arg Ala Gln Gly 80 85 90Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala Ala Gly Gly His 95 100 105Tyr (2) INFORMATION FOR SEQ ID NO: 83 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 106 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83 Leu LeuThr Leu Ser Pro His Tyr Lys Val Phe Leu Ala Arg Phe 1 5 10 15 Ile TrpTrp Leu Gln Tyr Leu Ile Thr Arg Thr Glu Ala His Leu 20 25 30 Gln Val TrpVal Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala 35 40 45 Ile Ile Leu LeuThr Cys Val Val His Pro Glu Leu Ile Phe Asp 50 55 60 Ile Thr Lys Leu LeuLeu Ala Ile Leu Gly Pro Leu Met Val Leu 65 70 75 Gln Ala Ser Ile Ile ArgVal Pro Tyr Phe Val Arg Ala Gln Gly 80 85 90 Leu Ile Arg Ala Cys Met LeuVal Arg Lys Val Ala Gly Gly His 95 100 105 Tyr (2) INFORMATION FOR SEQID NO: 84 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 84 Leu Leu Thr Leu Ser Pro His Tyr LysVal Phe Leu Ala Arg Phe 1 5 10 15 Val Trp Trp Leu Gln Tyr Leu Ile ThrArg Thr Glu Ala His Leu 20 25 30 Gln Val Trp Val Pro Pro Leu Asn Val ArgGly Gly Arg Asp Ala 35 40 45 Ile Thr Leu Leu Thr Cys Val Val His Pro GluLeu Ile Phe Asp 50 55 60 Ile Thr Lys Tyr Leu Leu Ala Ile Phe Gly Pro LeuMet Val Leu 65 70 75 Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg AlaGln Gly 80 85 90 Leu Ile Arg Ala Cys Met Leu Val Arg Lys Val Ala Gly GlyHis 95 100 105 Tyr (2) INFORMATION FOR SEQ ID NO: 85 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 92 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 85 Leu Phe Thr Leu Ser Pro His Tyr Lys Val Phe Leu Ala ArgLeu 1 5 10 15 Ile Trp Trp Leu Gln Tyr Phe Ile Thr Arg Ala Glu Ala HisLeu 20 25 30 Gln Val Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala35 40 45 Ile Ile Leu Leu Thr Cys Ala Val His Ser Glu Leu Ile Phe Asp 5055 60 Ile Thr Lys Ile Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu 65 7075 Gln Ala Gly Leu Thr Arg Val Pro Tyr Phe Val Ser Ala Gln Gly 80 85 90Leu Ile (2) INFORMATION FOR SEQ ID NO: 86 (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 106 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86 LeuLeu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu 1 5 10 15 IleTrp Trp Leu Gln Tyr Phe Ile Thr Arg Ala Glu Ala His Leu 20 25 30 Gln ValTrp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala 35 40 45 Ile Ile LeuLeu Ala Cys Ala Val His Pro Glu Pro Ile Phe Asp 50 55 60 Ile Thr Lys TyrLeu Leu Ala Ile Phe Gly Pro Leu Met Val Leu 65 70 75 Gln Ala Gly Ile ThrArg Val Pro Tyr Phe Trp Arg Ala Gln Gly 80 85 90 Leu Ile Arg Ala Cys MetLeu Ala Arg Lys Val Ala Gly Gly His 95 100 105 Tyr (2) INFORMATION FORSEQ ID NO: 87 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87 Leu Leu Thr Leu Ser Pro His TyrLys Val Phe Leu Ala Arg Leu 1 5 10 15 Met Trp Trp Leu Gln Tyr Phe LeuThr Arg Ala Glu Ala His Leu 20 25 30 Gln Val Trp Val Pro Ser Leu Asn ValArg Gly Gly Arg Asp Ala 35 40 45 Ile Ile Leu Leu Thr Cys Ala Val Tyr ProGlu Leu Ile Phe Asp 50 55 60 Ile Thr Lys Leu Leu Leu Ala Thr Leu Gly ProLeu Met Val Leu 65 70 75 Gln ala Gly Leu Thr Arg Val Pro Tyr Phe Val ArgAla Gln Gly 80 85 90 Leu Ile Arg Ala Cys Met Leu Val Arg Lys Val Val GlyGly His 95 100 105 Tyr (2) INFORMATION FOR SEQ ID NO: 88 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 106 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 88 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Leu Leu Ala ArgLeu 1 5 10 15 Ile Trp Trp Leu Gln Tyr Phe Ile Thr Arg Ala Glu Ala HisLeu 20 25 30 Gln Val Trp Ala Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala35 40 45 Ile Ile Leu Leu Met Cys Val Val His Pro Glu Leu Ile Phe Asp 5055 60 Ile Thr Lys Ile Leu Leu Ala Val Leu Gly Pro Leu Thr Val Leu 65 7075 Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Trp 80 85 90Leu Ile Arg Ala Cys Met Leu Val Arg Asn Ile Ala Gly Gly His 95 100 105Tyr (2) INFORMATION FOR SEQ ID NO: 89 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 106 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89 Leu LeuThr Leu Ser Pro His Tyr Lys Val Phe Leu Ala Ser Leu 1 5 10 15 Met TrpTrp Leu Gln Tyr Phe Leu Thr Arg Ala Glu Ala His Leu 20 25 30 Gln Val TrpVal Pro Ser Leu Asn Val Arg Gly Gly Arg Asp Ala 35 40 45 Ile Ile Leu LeuThr Cys Ala Val Tyr Pro Glu Leu Ile Leu Asp 50 55 60 Ile Thr Lys Leu LeuLeu Ala Ile Leu Gly Pro Leu Met Val Leu 65 70 75 Gln Ala Ser Ile Ile ArgVal Pro Tyr Phe Val Arg Ala Gln Gly 80 85 90 Leu Ile Arg Ala Cys Met LeuVal Arg Lys Ala Ala Gly Gly His 95 100 105 Tyr (2) INFORMATION FOR SEQID NO: 90 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 90 Leu Leu Thr Leu Ser Pro His Tyr LysVal Phe Leu Ala Arg Leu 1 5 10 15 Thr Trp Trp Leu Gln Tyr Phe Leu ThrArg Ala Glu Ala His Leu 20 25 30 Gln Val Trp Val Pro Ser Leu Asn Val ArgGly Gly Arg Asp Ala 35 40 45 Ile Ile Leu Leu Thr Cys Ala Val Tyr Pro GluLeu Ile Phe Asp 50 55 60 Ile Thr Lys Leu Leu Leu Ala Thr Leu Gly Pro LeuMet Val Leu 65 70 75 Gln Ala Gly Leu Thr Arg Val Pro Tyr Phe Val Arg AlaGln Gly 80 85 90 Leu Ile Arg Ala Cys Met Leu Val Arg Lys Val Ala Gly GlyHis 95 100 105 Tyr (2) INFORMATION FOR SEQ ID NO: 91 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 106 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 91 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala ArgLeu 1 5 10 15 Ile Trp Trp Leu Gln Tyr Phe Ile Thr Arg Ala Glu Ala HisLeu 20 25 30 Gln Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala35 40 45 Ile Ile Leu Leu Thr Cys Ala Val Tyr Pro Glu Leu Ile Phe Asp 5055 60 Ile Thr Lys Leu Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu 65 7075 Gln Ala Ser Ile Ile Arg Val Pro Tyr Phe Val Arg Ala Gln Gly 80 85 90Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala Ala Gly Val Asn 95 100 105Tyr (2) INFORMATION FOR SEQ ID NO: 92 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 106 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92 Leu PheThr Leu Ser Pro His Cys Lys Val Phe Leu Ala Arg Leu 1 5 10 15 Ile TrpTrp Leu Gln Tyr Phe Ile Thr Arg Ala Glu Ala His Leu 20 25 30 Gln Val TrpIle Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala 35 40 45 Ile Ile Leu LeuAla Cys Ala Val His Pro Glu Leu Ile Phe Asp 50 55 60 Ile Thr Lys Leu LeuLeu Ala Ile Leu Gly Pro Leu Met Val Leu 65 70 75 Gln Ala Ser Ile Ile ArgVal Pro Tyr Leu Tyr Arg Ala Gln Gly 80 85 90 Leu Ile Arg Ala Cys Met LeuVal Arg Lys Ala Ala Gly Gly His 95 100 105 Tyr (2) INFORMATION FOR SEQID NO: 93 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 93 Leu Phe Asn Leu Ser Pro His Tyr LysVal Phe Leu Ala Arg Leu 1 5 10 15 Ile Trp Trp Leu Gln Tyr Phe Ile ThrArg Ala Glu Ala His Leu 20 25 30 Gln Val Trp Ile Pro Pro Leu Asn Val GlnGly Gly Arg Asp Ala 35 40 45 Ile Ile Leu Leu Ala Cys Ala Val His Pro GluLeu Ile Phe Asp 50 55 60 Ile Thr Lys Leu Leu Leu Ala Ile Leu Gly Pro LeuMet Val Leu 65 70 75 Gln Ala Ser Ile Ile Arg Val Pro Tyr Phe Val Arg AlaGln Gly 80 85 90 Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala Ala Gly GlyHis 95 100 105 Tyr (2) INFORMATION FOR SEQ ID NO: 94 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 98 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 94 Ala Val Glu Phe Ile Pro Val Glu Ser Met Glu Thr Thr MetArg 1 5 10 15 Ser Pro Val Phe Thr Asp Asn Pro Ser Pro Pro Ala Val ProGln 20 25 30 Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys35 40 45 Ser Thr Arg Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val 5055 60 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala 65 7075 Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Leu Arg Thr Gly 80 85 90Val Arg Thr Ile Thr Thr Gly Ala 95 (2) INFORMATION FOR SEQ ID NO: 95 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 95 Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile ProIle Glu Ala Ile 1 5 10 15 Lys Gly Gly Arg His Leu Ile Phe Cys His SerLys Lys Lys Cys 20 25 30 Asp Glu Leu Ala Ala Lys Leu 35 (2) INFORMATIONFOR SEQ ID NO: 96 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9472 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION:KHCV-LBC1, Fig. 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96 TGCCAGCCCCCGATTGGGGG CGACACTCCA CCATAGATCA CTCCCCTGTG AGGAACTACT 60 GTCTTCACGCAGAAAGCGTC TAGCCATGGC GTTAGTATGA GTGTCGTGCA GCCTCCAGGA 120 CCCCCCCTCCCGGGAGAGCC ATAGTGGTCT GCGGAACCGG TGAGTACACC GGAATTGCCA 180 GGACGACCGGGTCCTTTCTT GGATCAACCC GCTCAATGCC TGGAGATTTG GGCGTGCCCC 240 CGCGAGACTGCTAGCCGAGT AGTGTTGGGT CGCGAAAGGC CTTGTGGTAC TGCCTGATAG 300 GGTGCTTGCGAGTGCCCCGG GAGGTCTCGT AGACCGTGCA CC ATG AGC ACG AAT 354 Met Ser Thr Asn1 CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC CGC CGC CCA CAG 402Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 5 10 1520 GAT ATT AAG TTC CCG GGC GGT GGT CAG ATC GTT GGT GGA GTT TAC TTG 450Asp Ile Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu 25 30 35TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCG ACT AGG AAG ACT 498 LeuPro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr 40 45 50 TCCGAG CGG TCG CAA CCT CGT GGA AGG CGA CAG CCT ATC CCC AAG GCT 546 Ser GluArg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala 55 60 65 CGC CGGCCC GAG GGC AGG GCC TGG GCT CAG CCC GGG TAC CCT TGG CCC 594 Arg Arg ProGlu Gly Arg Ala Trp Ala Gln Pro Gly Tyr Pro Trp Pro 70 75 80 CTC TAT GGCAAT GAG GGC TTG GGG TGG GCA GGA TGG CTC CTG TCA CCC 642 Leu Tyr Gly AsnGlu Gly Leu Gly Trp Ala Gly Trp Leu Leu Ser Pro 85 90 95 100 CGC GGC TCCCGG CCT AGT TGG GGC CCC ACG GAC CCC CGG CGT AAG TCG 690 Arg Gly Ser ArgPro Ser Trp Gly Pro Thr Asp Pro Arg Arg Lys Ser 105 110 115 CGT AAT TTGGGT AAG GTC ATC GAC ACC CTC ACA TGC GGC TTC GCC GAC 738 Arg Asn Leu GlyLys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp 120 125 130 CTC ATG GGGTAC ATT CCG CTC GTC GGC GCC CCC CTA GGG GGC GTT GCC 786 Leu Met Gly TyrIle Pro Leu Val Gly Ala Pro Leu Gly Gly Val Ala 135 140 145 AGG GCC CTGGCA CAT GGT GTC CGG GTG CTG GAG GAC GGC GTG AAC TAT 834 Arg Ala Leu AlaHis Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr 150 155 160 GCA ACA GGGAAT CTG CCC GGT TGC TCT TTC TCT ATC TTC CTC TTG GCT 882 Ala Thr Gly AsnLeu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala 165 170 175 180 CTG CTGTCT TGT TTG ACC ACC CCA GTT TCC GCT TAT GAA GTG CGT AAC 930 Leu Leu SerCys Leu Thr Thr Pro Val Ser Ala Tyr Glu Val Arg Asn 185 190 195 GCG TCCGGG ATG TAC CAT GTC ACG AAC GAC TGC TCC AAC TCA AGC ATT 978 Ala Ser GlyMet Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile 200 205 210 GTG TATGAG GCA GCG GAC ATG ATC ATG CAC ACT CCC GGG TGC GTG CCC 1026 Val Tyr GluAla Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro 215 220 225 TGC GTTCGG GAG GAC AAC TCC TCC CGT TGC TGG GTG GCA CTT ACT CCC 1074 Cys Val ArgGlu Asp Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro 230 235 240 ACG CTCGCG GCC AGG AAT GCC AGC GTC CCC ACT ACG ACA TTG CGA CGC 1122 Thr Leu AlaAla Arg Asn Ala Ser Val Pro Thr Thr Thr Leu Arg Arg 245 250 255 260 CATGTC GAC TTG CTC GTT GGG GTA GCT GCT TTC TGT TCC GCT ATG TAC 1170 His ValAsp Leu Leu Val Gly Val Ala Ala Phe Cys Ser Ala Met Tyr 265 270 275 GTGGGG GAC CTC TGC GGA TCT GTT TTC CTT GTT TCC CAG CTG TTC ACC 1218 Val GlyAsp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr 280 285 290 TTTTCG CCT CGC CGG CAT GAG ACG GTA CAG GAC TGC AAC TGC TCA ATC 1266 Phe SerPro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile 295 300 305 TATCCC GGC CGC GTA TCA GGT CAC CGC ATG GCC TGG GAT ATG ATG ATG 1314 Tyr ProGly Arg Val Ser Gly His Arg Met Ala Trp Asp Met Met Met 310 315 320 AACTGG TCG CCT ACA ACA GCC CTA GTG GTA TCG CAG CTA CTC CGG ATC 1362 Asn TrpSer Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile 325 330 335 340CCA CAA GCT GTC GTG GAC ATG GTG ACA GGG TCC CAC TGG GGA ATC CTG 1410 ProGln Ala Val Val Asp Met Val Thr Gly Ser His Trp Gly Ile Leu 345 350 355GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG GTC TTA 1458 AlaGly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu 360 365 370ATT GCG ATG CTA CTC TTT GCC GGC GTT GAC GGA ACC ACC CAC GTG ACA 1506 IleAla Met Leu Leu Phe Ala Gly Val Asp Gly Thr Thr His Val Thr 375 380 385GGG GGG GCG CAA GGT CGG GCC GCT AGC TCG CTA ACG TCC CTC TTT AGC 1554 GlyGly Ala Gln Gly Arg Ala Ala Ser Ser Leu Thr Ser Leu Phe Ser 390 395 400CCT GGG CCG GTT CAG CAC CTC CAG CTC ATA AAC ACC AAC GGC AGC TGG 1602 ProGly Pro Val Gln His Leu Gln Leu Ile Asn Thr Asn Gly Ser Trp 405 410 415420 CAT ATC AAC AGG ACC GCC CTG AGC TGC AAT GAC TCC CTC AAC ACT GGG 1650His Ile Asn Arg Thr Ala Leu Ser Cys Asn Asp Ser Leu Asn Thr Gly 425 430435 TTT GTT GCC GCG CTG TTC TAC AAA TAC AGG TTC AAC GCG TCC GGG TGC 1698Phe Val Ala Ala Leu Phe Tyr Lys Tyr Arg Phe Asn Ala Ser Gly Cys 440 445450 CCG GAG CGC TTG GCC ACG TGC CGC CCC ATT GAT ACA TTC GCG CAG GGG 1746Pro Glu Arg Leu Ala Thr Cys Arg Pro Ile Asp Thr Phe Ala Gln Gly 455 460465 TGG GGT CCC ATC ACT TAC ACT GAG CCT CAT GAT TTG GAT CAG AGG CCC 1794Trp Gly Pro Ile Thr Tyr Thr Glu Pro His Asp Leu Asp Gln Arg Pro 470 475480 TAT TGC TGG CAC TAC GCG CCT CAA CCG TGT GGT ATT GTG CCC ACG TTG 1842Tyr Cys Trp His Tyr Ala Pro Gln Pro Cys Gly Ile Val Pro Thr Leu 485 490495 500 CAG GTG TGT GGC CCA GTA TAC TGC TTC ACC CCG AGT CCT GTT GCG GTG1890 Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Ala Val 505510 515 GGG ACT ACC GAT CGT TTC GGT GCC CCT ACA TAC AGA TGG GGG GCA AAT1938 Gly Thr Thr Asp Arg Phe Gly Ala Pro Thr Tyr Arg Trp Gly Ala Asn 520525 530 GAG ACG GAC GTG CTG CTC CTT AAC AAC GCC GGG CCG CCG CAA GGC AAC1986 Glu Thr Asp Val Leu Leu Leu Asn Asn Ala Gly Pro Pro Gln Gly Asn 535540 545 TGG TTC GGC TGT ACA TGG ATG AAT GGC ACT GGG TTC ACC AAG ACA TGT2034 Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr Cys 550555 560 GGG GGC CCC CCG TGT AAC ATC GGG GGG GTC GGC AAC AAT ACC TTG ACC2082 Gly Gly Pro Pro Cys Asn Ile Gly Gly Val Gly Asn Asn Thr Leu Thr 565570 575 580 TGC CCC ACG GAC TGC TTC CGA AAG CAC CCC GGG GCC ACT TAC ACCAAA 2130 Cys Pro Thr Asp Cys Phe Arg Lys His Pro Gly Ala Thr Tyr Thr Lys585 590 595 TGC GGT TCG GGG CCT TGG TTA ACA CCC AGG TGC TTA GTC GAC TACCCG 2178 Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu Val Asp Tyr Pro600 605 610 TAC AGG CTC TGG CAT TAC CCC TGC ACT GTC AAC TTT ACC ATC TTTAAG 2226 Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe Lys615 620 625 GTT AGG ATG TAC GTG GGG GGC GCG GAG CAC AGG CTC GAC GCC GCATGC 2274 Val Arg Met Tyr Val Gly Gly Ala Glu His Arg Leu Asp Ala Ala Cys630 635 640 AAC TGG ACT CGG GGA GAG CGT TGT GAC CTG GAG GAC AGG GAT AGGTCA 2322 Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser645 650 655 660 GAG CTT AGC CCG CTG CTG CTG TCT ACA ACA GAG TGG CAG GTACTG CCC 2370 Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln Val LeuPro 665 670 675 TGT TCC TTC ACA ACC CTA CCG GCT CTG TCC ACT GGT TTG ATTCAT CTC 2418 Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile HisLeu 680 685 690 CAT CAG AAC ATC GTG GAC ATA CAA TAC CTG TAC GGT ATA GGGTCG GCG 2466 His Gln Asn Ile Val Asp Ile Gln Tyr Leu Tyr Gly Ile Gly SerAla 695 700 705 GTT GTC TCC TTT GCG ATC AAA TGG GAG TAT ATT GTG CTG CTCTTC CTT 2514 Val Val Ser Phe Ala Ile Lys Trp Glu Tyr Ile Val Leu Leu PheLeu 710 715 720 CTT CTG GCG GAC GCG CGC GTC TGC GCT TGC TTG TGG ATG ATGCTG CTG 2562 Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp Met Met LeuLeu 725 730 735 740 GTA GCG CAA GCC GAG GCC GCC TTA GAG AAC CTG GTG GTCCTC AAT GCA 2610 Val Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val LeuAsn Ala 745 750 755 GCG TCC GTG GCC GGA GCG CAT GGC ATT CTT TCC TTC ATTGTG TTC TTC 2658 Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Ile ValPhe Phe 760 765 770 TGT GCT GCC TGG TAC ATC AAG GGC AGG CTG GTT CCC GGAGCG GCA TAC 2706 Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly AlaAla Tyr 775 780 785 GCC CTC TAT GGC GTA TGG CCG CTG CTT CTG CTT CTG CTGGCG TTA CCA 2754 Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu AlaLeu Pro 790 795 800 CCA CGG GCG TAC GCC ATG GAC CGG GAG ATG GCC GCA TCGTGC GGA GGC 2802 Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser CysGly Gly 805 810 815 820 GCG GTT TTT GTA GGT CTG GTA CTC TTG ACC TTG TCACCA CAC TAT AAA 2850 Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser ProHis Tyr Lys 825 830 835 GTG TTC CTT GCC AGG TTC ATA TGG TGG CTA CAA TATCTC ATC ACC AGA 2898 Val Phe Leu Ala Arg Phe Ile Trp Trp Leu Gln Tyr LeuIle Thr Arg 840 845 850 ACC GAA GCG CAT CTG CAA GTG TGG GTC CCC CCT CTCAAC GTT CGG GGG 2946 Thr Glu Ala His Leu Gln Val Trp Val Pro Pro Leu AsnVal Arg Gly 855 860 865 GGT CGC GAT GCC ATC ATC CTC CTC ACA TGC GTG GTCCAC CCA GAG CTA 2994 Gly Arg Asp Ala Ile Ile Leu Leu Thr Cys Val Val HisPro Glu Leu 870 875 880 ATC TTT GAC ATC ACA AAA TAT TTG CTC GCC ATA TTCGGC CCG CTC ATG 3042 Ile Phe Asp Ile Thr Lys Tyr Leu Leu Ala Ile Phe GlyPro Leu Met 885 890 895 900 GTG CTC CAG GCC GGC ATA ACT AGA GTG CCG TACTTC GTG CGC GCA CAA 3090 Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr PheVal Arg Ala Gln 905 910 915 GGG CTC ATT CGT GCA TGC ATG TTG GCG CGG AAAGTC GTG GGG GGT CAT 3138 Gly Leu Ile Arg Ala Cys Met Leu Ala Arg Lys ValVal Gly Gly His 920 925 930 TAC GTC CAA ATG GTC TTC ATG AAG CTG GCC GCACTA GCA GGT ACG TAC 3186 Tyr Val Gln Met Val Phe Met Lys Leu Ala Ala LeuAla Gly Thr Tyr 935 940 945 GTT TAT GAC CAT CTT ACT CCA CTG CGA GAT TGGGCT CAC ACG GGC TTA 3234 Val Tyr Asp His Leu Thr Pro Leu Arg Asp Trp AlaHis Thr Gly Leu 950 955 960 CGA GAC CTT GCA GTG GCA GTA GAG CCC GTT GTCTTC TCT GAC ATG GAG 3282 Arg Asp Leu Ala Val Ala Val Glu Pro Val Val PheSer Asp Met Glu 965 970 975 980 ACC AAA GTC ATC ACC TGG GGG GCA GAC ACCGCG GCG TGC GGG GAC ATC 3330 Thr Lys Val Ile Thr Trp Gly Ala Asp Thr AlaAla Cys Gly Asp Ile 985 990 995 ATC TTG GCC TGC CCT GCT TCC GCC CGA AGGGGG AAG GAG ATA CTT CTG 3378 Ile Leu Ala Cys Pro Ala Ser Ala Arg Arg GlyLys Glu Ile Leu Leu 1000 1005 1010 GGA CCG GCC GAT AGT CTT GAA GGA CAGGGG TGG CGA CTC CTT GCG CCC 3426 Gly Pro Ala Asp Ser Leu Glu Gly Gln GlyTrp Arg Leu Leu Ala Pro 1015 1020 1025 ATC ACG GCC TAC TCC CAA CAA ACGCGA GGC CTG CTT GGT TGC ATC ATC 3474 Ile Thr Ala Tyr Ser Gln Gln Thr ArgGly Leu Leu Gly Cys Ile Ile 1030 1035 1040 ACT AGC CTT ACA GGC CGG GACAAG AAC CAG GTT GAG GGG GAG GTT CAA 3522 Thr Ser Leu Thr Gly Arg Asp LysAsn Gln Val Glu Gly Glu Val Gln 1045 1050 1055 1060 GTG GTT TCC ACC GCAACA CAA TCT TTC CTG GCG ACC TGC ATC AAT GGC 3570 Val Val Ser Thr Ala ThrGln Ser Phe Leu Ala Thr Cys Ile Asn Gly 1065 1070 1075 GTG TGT TGG ACTGTC TTC CAC GGC GCC GGC TCA AAG ACC CTA GCC GGC 3618 Val Cys Trp Thr ValPhe His Gly Ala Gly Ser Lys Thr Leu Ala Gly 1080 1085 1090 CCA AAG GGTCCA ATC ACC CAA ATG TAC ACC AAT GTA GAC CAG GAC CTT 3666 Pro Lys Gly ProIle Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu 1095 1100 1105 GTT GGCTGG CCG GCA CCT CCT GGG GCG CGT TCC CTG ACA CCA TGC ACT 3714 Val Gly TrpPro Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr 1110 1115 1120 TGCGGC TCC TCG GAC CTT TAC CTG GTC ACG AGA CAT GCT GAT GTC ATT 3762 Cys GlySer Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile 1125 1130 11351140 CCG GTG CGC CGG CGG GGT GAC GGT AGG GGG AGC CTA CTC CCC CCC AGG3810 Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Pro Pro Arg1145 1150 1155 CCT GTC TCC TAC TTG AAG GGC TCC TCG GGT GGT CCA CTG CTCTGC CCT 3858 Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu CysPro 1160 1165 1170 TCG GGG CAC GCT GTC GGC ATA CTT CCG GCT GCT GTA TGCACC CGG GGG 3906 Ser Gly His Ala Val Gly Ile Leu Pro Ala Ala Val Cys ThrArg Gly 1175 1180 1185 GTT GCC ATG GCG GTG GAA TTC ATA CCC GTT GAG TCTATG GAA ACT ACT 3954 Val Ala Met Ala Val Glu Phe Ile Pro Val Glu Ser MetGlu Thr Thr 1190 1195 1200 ATG CGG TCT CCG GTC TTC ACG GAC AAT CCG TCTCCC CCG GCT GTA CCG 4002 Met Arg Ser Pro Val Phe Thr Asp Asn Pro Ser ProPro Ala Val Pro 1205 1210 1215 1220 CAG ACA TTC CAA GTG GCC CAC TTA CACGCT CCC ACC GGC AGC GGC AAG 4050 Gln Thr Phe Gln Val Ala His Leu His AlaPro Thr Gly Ser Gly Lys 1225 1230 1235 AGC ACT AGG GTG CCG GCT GCA TATGCA GCC CAA GGG TAC AAG GTG CTC 4098 Ser Thr Arg Val Pro Ala Ala Tyr AlaAla Gln Gly Tyr Lys Val Leu 1240 1245 1250 GTC CTA AAT CCG TCC GTC GCCGCC ACC TTG GGT TTT GGG GCG TAT ATG 4146 Val Leu Asn Pro Ser Val Ala AlaThr Leu Gly Phe Gly Ala Tyr Met 1255 1260 1265 TCC AAG GCA CAT GGT ATCGAC CCC AAC CTT AGA ACT GGG GTA AGG ACC 4194 Ser Lys Ala His Gly Ile AspPro Asn Leu Arg Thr Gly Val Arg Thr 1270 1275 1280 ATC ACC ACA GGT GCCCCT ATC ACA TAC TCC ACC TAT GGC AAG TTC CTT 4242 Ile Thr Thr Gly Ala ProIle Thr Tyr Ser Thr Tyr Gly Lys Phe Leu 1285 1290 1295 1300 GCC GAC GGTGGC GGC TCC GGG GGC GCC TAT GAC ATC ATA ATG TGT GAT 4290 Ala Asp Gly GlyGly Ser Gly Gly Ala Tyr Asp Ile Ile Met Cys Asp 1305 1310 1315 GAG TGCCAC TCA ACT GAC TCG ACT ACC ATT TAT GGC ATC GGC ACA GTC 4338 Glu Cys HisSer Thr Asp Ser Thr Thr Ile Tyr Gly Ile Gly Thr Val 1320 1325 1330 CTGGAC CAA GCG GAG ACG GCT GGA GCG CGG CTC GTG GTG CTC TCC ACC 4386 Leu AspGln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ser Thr 1335 1340 1345GCT ACG CCT CCG GGA TCG GTC ACC GTG CCA CAC CTC AAT ATC GAG GAG 4434 AlaThr Pro Pro Gly Ser Val Thr Val Pro His Leu Asn Ile Glu Glu 1350 13551360 GTG GCC CTG TCT AAT ACT GGA GAG ATC CCC TTC TAC GGC AAA GCC ATT4482 Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile1365 1370 1375 1380 CCC ATC GAG GCT ATC AAG GGG GGA AGG CAT CTC ATT TTCTGC CAT TCC 4530 Pro Ile Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe CysHis Ser 1385 1390 1395 AAG AAG AAG TGT GAC GAA CTC GCC GCA AAG CTG TCAGGC CTC GGA CTC 4578 Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser GlyLeu Gly Leu 1400 1405 1410 AAT GCC GTA GCG TAT TAC CGG GGT CTT GAC GTGTCC GTC ATA CCG ACC 4626 Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val SerVal Ile Pro Thr 1415 1420 1425 AGC GGA GAC GTT GTT GTC GTG GCG ACG GACGCT CTA ATG ACG GGC TTT 4674 Ser Gly Asp Val Val Val Val Ala Thr Asp AlaLeu Met Thr Gly Phe 1430 1435 1440 ACC GGC GAC TTT GAC TCA GTG ATC GACTGT AAT ACG TGT GTC ACC CAG 4722 Thr Gly Asp Phe Asp Ser Val Ile Asp CysAsn Thr Cys Val Thr Gln 1445 1450 1455 1460 ACA GTC GAT TTC AGC TTG GACCCC ACC TTC ACC ATT GAG ACG ACG ACC 4770 Thr Val Asp Phe Ser Leu Asp ProThr Phe Thr Ile Glu Thr Thr Thr 1465 1470 1475 GTG CCC CAA GAC GCA GTGTCG CGC TCG CAG AGG CGA GGC AGG ACT GGT 4818 Val Pro Gln Asp Ala Val SerArg Ser Gln Arg Arg Gly Arg Thr Gly 1480 1485 1490 AGG GGC AGG GCT GGCATA TAC AGG TTT GTG ACT CCA GGA GAA CGG CCC 4866 Arg Gly Arg Ala Gly IleTyr Arg Phe Val Thr Pro Gly Glu Arg Pro 1495 1500 1505 TCG GGC ATG TTCGAT TCT TCG GTC CTG TGT GAG TGT TAT GAC GCG GGT 4914 Ser Gly Met Phe AspSer Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly 1510 1515 1520 TGT GCG TGGTAC GAA CTC ACG CCC GCT GAG ACC TCG GTT AGG TTG CGG 4962 Cys Ala Trp TyrGlu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg 1525 1530 1535 1540 GCGTAC CTA AAC ACA CCA GGG TTG CCC GTC TGC CAG GAC CAT CTG GAG 5010 Ala TyrLeu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu 1545 1550 1555TTC TCG GAG GGT GTC TTC ACA GGC CTC ACC CAC ATA GAT GCC CAC TTC 5058 PheSer Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe 1560 15651570 TTA TCC CAG ACT AAA CAG GCA GGA GAG AAC TTC CCC TAC TTG GTA GCA5106 Leu Ser Gln Thr Lys Gln Ala Gly Glu Asn Phe Pro Tyr Leu Val Ala1575 1580 1585 TAC CAG GCT ACA GTG TGC GCC AGG GCT CAA GCC CCA CCT CCATCG TGG 5154 Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro SerTrp 1590 1595 1600 GAT GAA ATG TGG AGG TGT CTC ATA CGG CTG AAA CCT ACGCTG CAC GGG 5202 Asp Glu Met Trp Arg Cys Leu Ile Arg Leu Lys Pro Thr LeuHis Gly 1605 1610 1615 1620 CCA ACA CCC CTG CTG TAT AGG TTA GGA GCC GTCCAA AAT GAG GTC ACC 5250 Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val GlnAsn Glu Val Thr 1625 1630 1635 CTC ACA CAC CCC ATA ACC AAA TTC ATC ATGACA TGT ATG TCG GCT GAC 5298 Leu Thr His Pro Ile Thr Lys Phe Ile Met ThrCys Met Ser Ala Asp 1640 1645 1650 CTG GAG GTC GTC ACC AGC ACC TGG GTGCTG GTA GGC GGA GTC CTC GCA 5346 Leu Glu Val Val Thr Ser Thr Trp Val LeuVal Gly Gly Val Leu Ala 1655 1660 1665 GCT CTG GCC GCG TAC TGC CTG ACAACA GGC AGC GTG GTC ATT GTG GGC 5394 Ala Leu Ala Ala Tyr Cys Leu Thr ThrGly Ser Val Val Ile Val Gly 1670 1675 1680 AGG ATC ATC CTG TCC GGG AAGCCG GCT ATC ATC CCC GAT AGG GAA GTT 5442 Arg Ile Ile Leu Ser Gly Lys ProAla Ile Ile Pro Asp Arg Glu Val 1685 1690 1695 1700 CTC TAC CAG GAG TTCGAC GAG ATG GAG GAG TGT GCC TCA CAC CTC CCT 5490 Leu Tyr Gln Glu Phe AspGlu Met Glu Glu Cys Ala Ser His Leu Pro 1705 1710 1715 TAC TTC GAA CAGGGA ATG CAG CTC GCC GAG CAA TTC AAA CAG AAG GCG 5538 Tyr Phe Glu Gln GlyMet Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala 1720 1725 1730 CTC GGG TTGCTG CAA ACA GCC ACC AAG CAG GCG GAG GCT GCT GCT CCC 5586 Leu Gly Leu LeuGln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro 1735 1740 1745 GTG GTGGAG TCC AAG TGG CGA GCC CTT GAG ACC TTC TGG GCG AAG CAC 5634 Val Val GluSer Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His 1750 1755 1760 ATGTGG AAC TTC ATT AGT GGG ATA CAG TAC TTG GCA GGC TTG TCC ACT 5682 Met TrpAsn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr 1765 1770 17751780 CTG CCT GGG AAC CCC GCA ATA CGA TCA CCG ATG GCA TTC ACA GCC TCC5730 Leu Pro Gly Asn Pro Ala Ile Arg Ser Pro Met Ala Phe Thr Ala Ser1785 1790 1795 ATC ACC AGC CCG CTC ACC ACC CAG CAT ACC CTC TTG TTT AACATC TTG 5778 Ile Thr Ser Pro Leu Thr Thr Gln His Thr Leu Leu Phe Asn IleLeu 1800 1805 1810 GGG GGA TGG GTG GCT GCC CAA CTC GCC CCC CCC AGC GCTGCC TCA GCT 5826 Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala AlaSer Ala 1815 1820 1825 TTC GTG GGC GCC GGC ATC GCT GGA GCC GCT GTT GGCACG ATA GGC CTT 5874 Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly ThrIle Gly Leu 1830 1835 1840 GGG AAG GTG CTT GTG GAC ATT CTG GCA GGT TATGGA GCA GGG GTG GCG 5922 Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr GlyAla Gly Val Ala 1845 1850 1855 1860 GGC GCA CTT GTG GCC TTT AAG ATC ATGAGC GGC GAG ATG CCT TCA GCC 5970 Gly Ala Leu Val Ala Phe Lys Ile Met SerGly Glu Met Pro Ser Ala 1865 1870 1875 GAG GAC ATG GTC AAC TTA CTC CCTGCC ATC CTT TCT CCC GGT GCC CTG 6018 Glu Asp Met Val Asn Leu Leu Pro AlaIle Leu Ser Pro Gly Ala Leu 1880 1885 1890 GTC GTC GGG ATT GTG TGT GCAGCA ATA CTG CGT CGG CAT GTG GGC CCA 6066 Val Val Gly Ile Val Cys Ala AlaIle Leu Arg Arg His Val Gly Pro 1895 1900 1905 GGG GAA GGG GCT GTG CAGTGG ATG AAC CGG CTG ATA GCG TTC GCC TCG 6114 Gly Glu Gly Ala Val Gln TrpMet Asn Arg Leu Ile Ala Phe Ala Ser 1910 1915 1920 CGG GGT AAC CAC GTCTCC CCC AGG CAC TAT GTG CCA GAG AGC GAG CCT 6162 Arg Gly Asn His Val SerPro Arg His Tyr Val Pro Glu Ser Glu Pro 1925 1930 1935 1940 GCA GCG CGTGTT ACC CAG ATC CTT TCC AGC CTC ACC ATC ACT CAG CTG 6210 Ala Ala Arg ValThr Gln Ile Leu Ser Ser Leu Thr Ile Thr Gln Leu 1945 1950 1955 TTG AAGAGA CTC CAC CAG TGG ATT AAT GAG GAC TGC TCT ACG CCA TGC 6258 Leu Lys ArgLeu His Gln Trp Ile Asn Glu Asp Cys Ser Thr Pro Cys 1960 1965 1970 TCCAGC TCG TGG CTA AGG GAG ATT TGG GAC TGG ATC TGC ACG GTG TTG 6306 Ser SerSer Trp Leu Arg Glu Ile Trp Asp Trp Ile Cys Thr Val Leu 1975 1980 1985ACT GAC TTC AAG ACC TGG CTC CAG TCC AAG CTC CTG CCG CGA TTA CCG 6354 ThrAsp Phe Lys Thr Trp Leu Gln Ser Lys Leu Leu Pro Arg Leu Pro 1990 19952000 GGA GTC CCT TTT TTC TCA TGC CAA CGC GGG TAT AAG GGA GTC TGG CGG6402 Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg2005 2010 2015 2020 GGG GAC GGC ATC ATG CAC ACC ACC TGC CCA TGC GGA GCACAG ATC ACC 6450 Gly Asp Gly Ile Met His Thr Thr Cys Pro Cys Gly Ala GlnIle Thr 2025 2030 2035 GGA CAC GTC AAA AAC GGT TCC ATG AGG ATC GTT GGGCCT AAA ACC TGC 6498 Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly ProLys Thr Cys 2040 2045 2050 AGC AAC ACG TGG TAC GGG ACA TTC CCC ATC AACGCG TAC ACC ACG GGC 6546 Ser Asn Thr Trp Tyr Gly Thr Phe Pro Ile Asn AlaTyr Thr Thr Gly 2055 2060 2065 CCC TGC ACA CCC TCC CCG GCG CCA AAC TATTCC AAG GCA TTG TGG AGA 6594 Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr SerLys Ala Leu Trp Arg 2070 2075 2080 GTG GCC GCT GAG GAG TAC GTG GAG GTCACG CGG GTG GGA GAT TTT CAC 6642 Val Ala Ala Glu Glu Tyr Val Glu Val ThrArg Val Gly Asp Phe His 2085 2090 2095 2100 TAC GTG ACG GGC ATG ACC ACTGAC AAC GTG AAG TGT CCA TGC CAG GTT 6690 Tyr Val Thr Gly Met Thr Thr AspAsn Val Lys Cys Pro Cys Gln Val 2105 2110 2115 CCG GCC CCC GAA TTC TTCACG GAG GTG GAT GGA GTG CGG TTG CAC AGG 6738 Pro Ala Pro Glu Phe Phe ThrGlu Val Asp Gly Val Arg Leu His Arg 2120 2125 2130 TAC GCT CCG GCG TGCAGA CCT CTC CTA CGG GAG GAG GTC GTA TTC CAG 6786 Tyr Ala Pro Ala Cys ArgPro Leu Leu Arg Glu Glu Val Val Phe Gln 2135 2140 2145 GTC GGG CTC CACCAG TAC CTG GTC GGG TCA CAG CTC CCA TGC GAG CCC 6834 Val Gly Leu His GlnTyr Leu Val Gly Ser Gln Leu Pro Cys Glu Pro 2150 2155 2160 GAA CCG GATGTA GCA GTG CTC ACT TCC ATG CTC ACT GAC CCC TCC CAC 6882 Glu Pro Asp ValAla Val Leu Thr Ser Met Leu Thr Asp Pro Ser His 2165 2170 2175 2180 ATTACA GCA GAG ACG GCT AAG CGT AGG CTG GCC AGG GGG TCT CCC CCC 6930 Ile ThrAla Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro Pro 2185 2190 2195TCC TTG GCC AGC TCT TCA GCT AGC CAG TTG TCT GCG CCT TCC TTG AAG 6978 SerLeu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys 2200 22052210 GCG ACA TGC ACT ACC CAT CAT GAC TCC CCG GAC GCT GAC CTC ATT GAG7026 Ala Thr Cys Thr Thr His His Asp Ser Pro Asp Ala Asp Leu Ile Glu2215 2220 2225 GCC AAC CTC TTG TGG CGG CAA GAG ATG GGC GGG AAC ATC ACCCGC GTG 7074 Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr ArgVal 2230 2235 2240 GAG TCA GAG AAT AAG GTG GTA ATC CTG GAC TCT TTC GACCCG CTC CGA 7122 Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp ProLeu Arg 2245 2250 2255 2260 GCG GAG GAT GAT GAG GGG GAA ATA TCC GTT CCGGCG GAG ATC CTG CGG 7170 Ala Glu Asp Asp Glu Gly Glu Ile Ser Val Pro AlaGlu Ile Leu Arg 2265 2270 2275 AAA TCC AGG AAA TTC CCC CCA GCG CTG CCCATA TGG GCG CCG CCG GAT 7218 Lys Ser Arg Lys Phe Pro Pro Ala Leu Pro IleTrp Ala Pro Pro Asp 2280 2285 2290 TAC AAC CCT CCG CTG CTA GAG TCC TGGAAG GAC CCG GAC TAC GTT CCT 7266 Tyr Asn Pro Pro Leu Leu Glu Ser Trp LysAsp Pro Asp Tyr Val Pro 2295 2300 2305 CCG GTG GTA CAC GGG TGC CCG TTGCCG CCC ACC AAG GCC CCT CCA ATA 7314 Pro Val Val His Gly Cys Pro Leu ProPro Thr Lys Ala Pro Pro Ile 2310 2315 2320 CCA CCT CCA CGG AGG AAG AGGACG GTT GTC CTG ACA GAA TCC ACC GTG 7362 Pro Pro Pro Arg Arg Lys Arg ThrVal Val Leu Thr Glu Ser Thr Val 2325 2330 2335 2340 TCT TCT GCC TTG GCGGAG CTC GCT ACT AAG ACC TTC GGC AGC TCC GGA 7410 Ser Ser Ala Leu Ala GluLeu Ala Thr Lys Thr Phe Gly Ser Ser Gly 2345 2350 2355 TCG TCG GCC ATCGAC AGC GGT ACG GCG ACC GCC CCT CCT GAC CAA GCC 7458 Ser Ser Ala Ile AspSer Gly Thr Ala Thr Ala Pro Pro Asp Gln Ala 2360 2365 2370 TCC GGT GACGGC GAC AGA GAG TCC GAC GTT GAG TCG TTC TCC TCC ATG 7506 Ser Gly Asp GlyAsp Arg Glu Ser Asp Val Glu Ser Phe Ser Ser Met 2375 2380 2385 CCC CCCCTT GAG GGA GAG CCG GGG GAC CCC GAT CTC AGC GAC GGA TCT 7554 Pro Pro LeuGlu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser 2390 2395 2400 TGGTCC ACC GTG AGC GAG GAG GCT AGT GAG GAC GTC GTC TGC TGT TCG 7602 Trp SerThr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser 2405 2410 24152420 ATG TCC TAC ACA TGG ACA GGC GCC CTG ATC ACG CCA TGC GCT GCG GAG7650 Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala Glu2425 2430 2435 GAA AGC AAG TTG CCC ATC AAC CCG TTG AGC AAT TCT TTG CTACGT CAC 7698 Glu Ser Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu ArgHis 2440 2445 2450 CAC AAC ATG GTC TAT GCT ACA ACA TCC CGC AGC GCA GGCCTG CGG CAG 7746 His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Gly LeuArg Gln 2455 2460 2465 AAG AAG GTC ACC TTT GAC AGA CTG CAA GTC CTG GACGAC CAC TAC CGG 7794 Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp AspHis Tyr Arg 2470 2475 2480 GAC GTG CTT AAG GAG ATG AAG GCG AAG GCG TCCACA GTT AAG GCT AAA 7842 Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser ThrVal Lys Ala Lys 2485 2490 2495 2500 CTT CTA TCT GTA GAA GAA GCC TGC AAACTG ACG CCC CCA CAT TCG GCC 7890 Leu Leu Ser Val Glu Glu Ala Cys Lys LeuThr Pro Pro His Ser Ala 2505 2510 2515 AAA TCC AAA TTT GGC TAC GGG GCGAAG GAC GTC CGG AGC CTA TCC AGC 7938 Lys Ser Lys Phe Gly Tyr Gly Ala LysAsp Val Arg Ser Leu Ser Ser 2520 2525 2530 AGG GCC GTT ACC CAC ATC CGCTCC GTG TGG AAG GAC CTG CTG GAA GAC 7986 Arg Ala Val Thr His Ile Arg SerVal Trp Lys Asp Leu Leu Glu Asp 2535 2540 2545 ACT GAA ACA CCA ATT AGCACT ACC ATC ATG GCA AAA AAT GAG GTT TTC 8034 Thr Glu Thr Pro Ile Ser ThrThr Ile Met Ala Lys Asn Glu Val Phe 2550 2555 2560 TGT GTC CAA CCA GAGAAG GGA GGC CGC AAG CCA GCT CGC CTT ATC GTG 8082 Cys Val Gln Pro Glu LysGly Gly Arg Lys Pro Ala Arg Leu Ile Val 2565 2570 2575 2580 TTC CCA GATCTG GGA GTT CGT GTA TGC GAG AAG ATG GCC CTT TAT GAC 8130 Phe Pro Asp LeuGly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp 2585 2590 2595 GTG GTCTCC ACC CTT CCT CAG GCC GTG ATG GGC TCC TCA TAC GGA TTC 8178 Val Val SerThr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly Phe 2600 2605 2610 CAGTAC TCT CCT AAG CAG CGG GTC GAG TTC CTG GTG AAT ACC TGG AAA 8226 Gln TyrSer Pro Lys Gln Arg Val Glu Phe Leu Val Asn Thr Trp Lys 2615 2620 2625TCA AAG AAA TGC CCC ATG GGC TTC TCA TAT GAC ACC CGC TGT TTT GAC 8274 SerLys Lys Cys Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp 2630 26352640 TCA ACG GTC ACT GAG AAT GAC ATC CGT GTT GAG GAG TCA ATT TAC CAA8322 Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile Tyr Gln2645 2650 2655 2660 TGT TGT GAC TTG GCC CCC GAA GCC AAA CTG GCC ATA AAGTCG CTC ACA 8370 Cys Cys Asp Leu Ala Pro Glu Ala Lys Leu Ala Ile Lys SerLeu Thr 2665 2670 2675 GAG CGG CTC TAT ATC GGG GGT CCC CTG ACT AAT TCAAAA GGG CAG AAC 8418 Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser LysGly Gln Asn 2680 2685 2690 TGC GGT TAC CGC CGG TGC CGC GCG AGC GGC GTGCTG ACG ACT AGC TGC 8466 Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val LeuThr Thr Ser Cys 2695 2700 2705 GGT AAT ACC CTC ACA TGT TAC CTG AAA GCCACT GCG GCC TGT CGA GCT 8514 Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala ThrAla Ala Cys Arg Ala 2710 2715 2720 GCG AAG CTC CGG GAC TGC ACG ATG CTCGTG AAC GGA GAC GAC CTT GTC 8562 Ala Lys Leu Arg Asp Cys Thr Met Leu ValAsn Gly Asp Asp Leu Val 2725 2730 2735 2740 GTT ATC TGT GAA AGC GCG GGAACC CAA GAG GAT GCG GCG AGC CTA CGA 8610 Val Ile Cys Glu Ser Ala Gly ThrGln Glu Asp Ala Ala Ser Leu Arg 2745 2750 2755 GTC TTC ACG GAG GCT ATGACT AGG TAC TCT GCC CCC CCT GGG GAC CCG 8658 Val Phe Thr Glu Ala Met ThrArg Tyr Ser Ala Pro Pro Gly Asp Pro 2760 2765 2770 CCT CAA CCG GAA TACGAC TTG GAG TTG ATA ACA TCA TGT TCC TCC AAT 8706 Pro Gln Pro Glu Tyr AspLeu Glu Leu Ile Thr Ser Cys Ser Ser Asn 2775 2780 2785 GTG TCG GTC GCACAC GAT GCA TCT GGT AAA AGG GTG TAC TAC CTC ACC 8754 Val Ser Val Ala HisAsp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr 2790 2795 2800 CGT GAC CCTACC ACC CCC CTT GCA CGG GCT GCG TGG GAG ACA GCT AGA 8802 Arg Asp Pro ThrThr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg 2805 2810 2815 2820 CACACT CCA GTC AAC TCC TGG CTA GGC AAC ATC ATC ATG TAT GCG CCC 8850 His ThrPro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro 2825 2830 2835ACC TTA TGG GCA AGG ATG ATT CTG ATG ACT CAT TTC TTC TCC ATC CTT 8898 ThrLeu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu 2840 28452850 CTA GCT CAG GAG CAA CTT GAA AAA ACC CTA GAT TGT CAG ATC TAC GGG8946 Leu Ala Gln Glu Gln Leu Glu Lys Thr Leu Asp Cys Gln Ile Tyr Gly2855 2860 2865 GCC TGT TAC TCC ATT GAA CCA CTT GAT CTA CCT CAG ATC ATTGAG CGA 8994 Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile GluArg 2870 2875 2880 CTC CAT GGT CTT AGC GCA TTT TCA CTC CAT AGT TAC TCTCCA GGC GAG 9042 Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser ProGly Glu 2885 2890 2895 2900 ATC AAT AGG GTG GCT TCA TGC CTC AGA AAA CTTGGG GTA CCA CCC TTG 9090 Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu GlyVal Pro Pro Leu 2905 2910 2915 CGA GCC TGG AGA CAT CGG GCC AGA AGT GTCCGC GCT AAG CTA CTG TCC 9138 Arg Ala Trp Arg His Arg Ala Arg Ser Val ArgAla Lys Leu Leu Ser 2920 2925 2930 CAG GGG GGG AGG GCC GCC ACT TGT GGCAAG TAC CTC TTC AAC TGG GCG 9186 Gln Gly Gly Arg Ala Ala Thr Cys Gly LysTyr Leu Phe Asn Trp Ala 2935 2940 2945 GTG AGG ACC AAG CTC AAA CTC ACTCCA ATC CCA GCC GCG TCC CGG TTG 9234 Val Arg Thr Lys Leu Lys Leu Thr ProIle Pro Ala Ala Ser Arg Leu 2950 2955 2960 GAC TTG TCC GGC TGG TTC GTTGCT GGT TAC AGC GGG GGA GAC ATA TAT 9282 Asp Leu Ser Gly Trp Phe Val AlaGly Tyr Ser Gly Gly Asp Ile Tyr 2965 2970 2975 2980 CAC AGC CTG TCT CGTGCC CGA CCC CGC TGG TTC ATG TTG TGC CTA CTC 9330 His Ser Leu Ser Arg AlaArg Pro Arg Trp Phe Met Leu Cys Leu Leu 2985 2990 2995 CTA CTT TCC GTGGGG GTA GGC ATC TAC CTG CTC CCC AAC CGA TGAATGG 9380 Leu Leu Ser Val GlyVal Gly Ile Tyr Leu Leu Pro Asn Arg 3000 3005 3010 GAGCTAAACA CTCCAGGCCAATAGGCCGTT TCTCTTTTTT TTTTTTTTTT TTTTTTTTTT 9440 TTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TT 9472 (2) INFORMATION FOR SEQ ID NO: 97 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 277 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: NS2-LBC2, Fig. 7 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 97 CTC TTT ACC CTG TCA CCA CAC TAC AAA GTG TTCCTC GCT AGG CTC ATA 48 Leu Phe Thr Leu Ser Pro His Tyr Lys Val Phe LeuAla Arg Leu Ile 1 5 10 15 TGG TGG TTA CAG TAT TTT ATC ACC AGG GCC GAAGCG CAC CTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Ile Thr Arg Ala Glu AlaHis Leu Gln Val 20 25 30 TGG ATC CCC CCC CTC AAC GTT CGG GGG GGC CGC GATGCC ATC ATC CTC 144 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp AlaIle Ile Leu 35 40 45 CTC ACG TGT GCG GTC CAC TCA GAG CTG ATT TTT GAC ATCACC AAG ATC 192 Leu Thr Cys Ala Val His Ser Glu Leu Ile Phe Asp Ile ThrLys Ile 50 55 60 TTG CTC GCC ATA CTT GGT CCG CTC ATG GTA CTC CAG GCT GGCCTA ACC 240 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly LeuThr 65 70 75 80 AGA GTG CCG TAC TTT GTC AGC GCT CAA GGG CTC ATC C 277Arg Val Pro Tyr Phe Val Ser Ala Gln Gly Leu Ile 85 90 (2) INFORMATIONFOR SEQ ID NO: 98 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 360 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION:KHCV366 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98 GGCCAGCCCC CGATTGGGGGCGACACTCCA CCATAGATCA CTCCCCTGTG AGGAACTACT 60 GTCTTCACGC AGAAAGCGTCTAGCCATGGC GTTAGTATGA GTGTCGTGCA GCCTCCAGGA 120 CCCCCCCTCC CGGGAGAGCCATAGTGGTCT GCGGAACCGG TGAGTACACC GGAATTGCCA 180 GGACGACCGG GTCCTTTCTTGGATCAACCC GCTCAATGCC TGGAGATTTG GGCGTGCCCC 240 CGCGAGACTG CTAGCCGAGTAGTGTTGGGT CGCGAAAGGC CTTGTGGTAC TGCCTGATAG 300 GGTGCTTGCG AGTGCCCCGGGAGGTCTCGT AGACCGTGCA CC ATG AGC ACG AAT 354 Met Ser Thr Asn 1 CCT AAA360 Pro Lys 5 (2) INFORMATION FOR SEQ ID NO: 99 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 359 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: HCPT-CHIRON, Fig 6 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 99 GCCAGCCCCC TGATGGGGGC GACACTCCAC CATGAATCACTCCCCTGTGA GGAACTACTG 60 TCTTCACGCA GAAAGCGTCT AGCCATGGCG TTAGTATGAGTGTCGTGCAG CCTCCAGGAC 120 CCCCCCTCCC GGGAGAGCCA TAGTGGTCTG CGGAACCGGTGAGTACACCG GAATTGCCAG 180 GACGACCGGG TCCTTTCTTG GATCAACCCG CTCAATGCCTGGAGATTTGG GCGTGCCCCC 240 GCAAGACTGC TAGCCGAGTA GTGTTGGGTC GCGAAAGGCCTTGTGGTACT GCCTGATAGG 300 GTGCTTGCGA GTGCCCCGGG AGGTCTCGTA GACCGTGCAC CATG AGC ACG AAT CCT 356 Met Ser Thr Asn Pro 1 5 AAA 359 Lys (2)INFORMATION FOR SEQ ID NO: 100 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:347 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: JHCV-NCI, Fig 6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100TTGGGGGCGA CACTCCACCA TAGATCACTC CCCTGTGAGG AACTACTGTC TTCACGCAGA 60AAGCGTCTAG CCATGGCGTT AGTATGAGTG TTGTGCAGCC TCCAGGACCC CCCCTCCCGG 120GAGAGCCATA GTGGTCTGCG GAACCGGTGA GTACACCGGA ATTGCCAGGA CGACCGGGTC 180CTTTCTTGGA TCAACGCGCT CAATGCCTGG AGATTTGGGC GTGCCCCCGC GAGACTGCTA 240GCCGAGTAGT GTTGGGTCGC GAAAGGCCTT GTGGTACTGC CTGATAGGGT GCTTGCGAGT 300GCCCCGGGAG GTCTCGTAGA CCGTGCATC ATG AGC ACA AAT CCT AAA 347 Met Ser ThrAsn Pro Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 101 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: NS2-LBC3, Fig.8 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 101 CTC TTG ACC TTG TCA CCA TAC TAT AAA GTG TTCCTC GCT AGG CTC ATA 48 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe LeuAla Arg Leu Ile 1 5 10 15 TGG TGG TTG CAA TAT TTT ATC ACC AGA GCC GAGGCG CAC TTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Ile Thr Arg Ala Glu AlaHis Leu Gln Val 20 25 30 TGG ATC CCC CCT CTC AAC GTC CGG GGA GGC CGT GATGCA ATC ATC CTC 144 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp AlaIle Ile Leu 35 40 45 CTG GCG TGT GCG GTC CAC CCA GAG CCG ATC TTT GAC ATCACA AAA TAT 192 Leu Ala Cys Ala Val His Pro Glu Pro Ile Phe Asp Ile ThrLys Tyr 50 55 60 TTG CTC GCC ATA TTC GGC CCG CTC ATG GTG CTC CAG GCC GGCATA ACT 240 Leu Leu Ala Ile Phe Gly Pro Leu Met Val Leu Gln Ala Gly IleThr 65 70 75 80 AGA GTG CCG TAC TTC TGG CGC GCA CAA GGG CTC ATT CGT GCATGC ATG 288 Arg Val Pro Tyr Phe Trp Arg Ala Gln Gly Leu Ile Arg Ala CysMet 85 90 95 TTG GCG CGG AAA GTC GCT GGG GGT CAT TAC 318 Leu Ala Arg LysVal Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO: 102 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 315 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC20, Fig. 9 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 102 CTC TTG ACC TTG TCA CCA CAC TAT AAA GTG TTCCTT GCC AGG TTC ATA 48 Leu Leu Thr Leu Ser Pro His Tyr Lys Val Phe LeuAla Arg Phe Ile 1 5 10 15 TGG TGG CTA CAA TAT CTC ATC ACC AGA ACC GAAGCG CAT CTG CAA GTG 96 Trp Trp Leu Gln Tyr Leu Ile Thr Arg Thr Glu AlaHis Leu Gln Val 20 25 30 TGG GTC CCC CCT CTC AAC GTT CGA GGA GGC CGT GATGCC GTC ATC CTC 144 Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp AlaVal Ile Leu 35 40 45 CTC ACG TGC GCA GTC TAC CCA GAG CTA ATC TTT GAC ATCACC AAA CTC 192 Leu Thr Cys Ala Val Tyr Pro Glu Leu Ile Phe Asp Ile ThrLys Leu 50 55 60 CTG CTT GCC ACA CTC GGT CCG CTC ATG GTG CTC CAG GCT GGCTTA ATT 240 Leu Leu Ala Thr Leu Gly Pro Leu Met Val Leu Gln Ala Gly LeuIle 65 70 75 80 AGA GTG CCG TAC TTC GTA CGC TCA GGG CTC ATT CGT GCA TGCATG TTG 288 Arg Val Pro Tyr Phe Val Arg Ser Gly Leu Ile Arg Ala Cys MetLeu 85 90 95 GTG CGG AAA GTT GCT GGG GGT CAT TAT 315 Val Arg Lys Val AlaGly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO: 103 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: NS2-LBC21, Fig. 10 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 103 CTC TTG ACC CTG TCA CCA CAC TAT AAA GTG TTCCTC GCT AGG CTC ATG 48 Leu Leu Thr Leu Ser Pro His Tyr Lys Val Phe LeuAla Arg Leu Met 1 5 10 15 TGG TGG TTA CAA TAC TTC CTC ACC AGA GCC GAAGCG CAC TTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Leu Thr Arg Ala Glu AlaHis Leu Gln Val 20 25 30 TGG GTC CCC TCT CTC AAC GTT CGA GGA GGC CGC GATGCC ATC ATC CTC 144 Trp Val Pro Ser Leu Asn Val Arg Gly Gly Arg Asp AlaIle Ile Leu 35 40 45 CTC ACG TGC GCA GTC TAC CCA GAG CTA ATC TTT GAC ATCACC AAA CTC 192 Leu Thr Cys Ala Val Tyr Pro Glu Leu Ile Phe Asp Ile ThrLys Leu 50 55 60 TTG CTT GCC ACA CTC GGC CCG CTC ATG GTG CTC CAG GCT GGCTTA ACT 240 Leu Leu Ala Thr Leu Gly Pro Leu Met Val Leu Gln Ala Gly LeuThr 65 70 75 80 AGA GTG CCG TAC TTT GTG CGC GCC CAG GGG CTC ATT CGT GCGTGC ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala CysMet 85 90 95 TTG GTG CGG AAA GTT GTG GGG GGC CAT TAT 318 Leu Val Arg LysVal Val Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO: 104 (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC23, Fig. 11 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 104 CTC TTG ACC TTG TCA CCA CAC TAT AAAGTG TTC CTT GCC AGG TTC ATA 48 Leu Leu Thr Leu Ser Pro His Tyr Lys ValPhe Leu Ala Arg Phe Ile 1 5 10 15 TGG TGG CTA CAA TAT CTC ATC ACC AGAACC GAA GCG CAT CTG CAA GTG 96 Trp Trp Leu Gln Tyr Leu Ile Thr Arg ThrGlu Ala His Leu Gln Val 20 25 30 TGG GTC CCC CCT CTC AAC GTT CGG GGG GGTCGC GAT GCC ATC ATC CTC 144 Trp Val Pro Pro Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC GCG TGT GCG GTC CAC CCA GAG CTG ATC TTTGAC ATC ACC AAA CTC 192 Leu Ala Cys Ala Val His Pro Glu Leu Ile Phe AspIle Thr Lys Leu 50 55 60 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTG CTC CAGGCT AGC ATA ATT 240 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln AlaSer Ile Ile 65 70 75 80 CGA GTG CCG TAC TCC GTG CGC GCT CAA GGC CTC ATTCGT GCA TGC ATG 288 Arg Val Pro Tyr Ser Val Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GCC GCC GGG GGT CAT TAT 318 Leu ValArg Lys Ala Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:105 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC25, Fig. 12 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 105 CTC TTG ACC TTG TCA CCA TAC TAT AAGGTG CTC CTC GCT AGG CTC ATA 48 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys ValLeu Leu Ala Arg Leu Ile 1 5 10 15 TGG TGG TTG CAA TAT TTT ATC ACC AGAGCC GAG GCG CAC TTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Ile Thr Arg AlaGlu Ala His Leu Gln Val 20 25 30 TGG GCT CCC CCC CTT AAC GTT CGG GGG GGCCGC GAT GCC ATC ATC CTC 144 Trp Ala Pro Pro Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC ATG TGT GTA GTT CAC CCG GAG CTA ATC TTTGAC ATC ACA AAA ATC 192 Leu Met Cys Val Val His Pro Glu Leu Ile Phe AspIle Thr Lys Ile 50 55 60 CTG CTC GCC GTG CTC GGT CCG CTC ACG GTG CTC CAGGCT GGC ATA ACC 240 Leu Leu Ala Val Leu Gly Pro Leu Thr Val Leu Gln AlaGly Ile Thr 65 70 75 80 CGA GTG CCG TAC TTT GTG CGC GCT CAA TGG CTC ATTCGT GCG TGC ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Trp Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAC ATC GCT GGG GGT CAT TAT 318 Leu ValArg Asn Ile Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:106 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC26, Fig. 13 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 106 CTC TTG ACC TTG TCA CCA CAC TAT AAAGTG TTC CTT GCC AGG TTC ATA 48 Leu Leu Thr Leu Ser Pro His Tyr Lys ValPhe Leu Ala Arg Phe Ile 1 5 10 15 TGG TGG CTA CAA TAT CTC ATC ACC AGAACC GAA GCG CAT CTG CAA GTG 96 Trp Trp Leu Gln Tyr Leu Ile Thr Arg ThrGlu Ala His Leu Gln Val 20 25 30 TGG GTC CCC CCT CTC AAC GTT CGG GGG GGTCGC GAT GCC ATC ATC CTC 144 Trp Val Pro Pro Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC ACA TGC GTG GTC CAC CCA GAG CTA ATC TTTGAC ATC ACC AAA CTC 192 Leu Thr Cys Val Val His Pro Glu Leu Ile Phe AspIle Thr Lys Leu 50 55 60 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTG CTC CAGGCT AGC ATA ATT 240 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln AlaSer Ile Ile 65 70 75 80 CGA GTG CCG TAC TTT GTG CGC GCT CAA GGC CTC ATTCGT GCA TGT ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GTT GCT GGG GGT CAT TAT 318 Leu ValArg Lys Val Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:107 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC27, Fig. 14 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 107 CTC TTG ACT CTG TCG CCA CAC TAT AAAGTG TTC CTC GCT AGC CTC ATG 48 Leu Leu Thr Leu Ser Pro His Tyr Lys ValPhe Leu Ala Ser Leu Met 1 5 10 15 TGG TGG TTA CAA TAC TTC CTC ACC AGAGCC GAA GCG CAC TTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Leu Thr Arg AlaGlu Ala His Leu Gln Val 20 25 30 TGG GTC CCC TCT CTC AAC GTT CGA GGA GGCCGC GAT GCC ATC ATC CTC 144 Trp Val Pro Ser Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC ACG TGC GCA GTC TAC CCA GAG CTA ATC TTAGAC ATC ACC AAA CTC 192 Leu Thr Cys Ala Val Tyr Pro Glu Leu Ile Leu AspIle Thr Lys Leu 50 55 60 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTG CTC CAGGCT AGC ATA ATT 240 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln AlaSer Ile Ile 65 70 75 80 CGA GTG CCG TAC TTC GTA CGC GCT CAA GGC CTC ATTCGT GCA TGC ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GCC GCC GGG GGT CAT TAT 318 Leu ValArg Lys Ala Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:108 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC28, Fig. 15 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 108 CTC TTG ACC CTG TCA CCG CAC TAT AAAGTG TTC CTC GCT AGG CTC ACG 48 Leu Leu Thr Leu Ser Pro His Tyr Lys ValPhe Leu Ala Arg Leu Thr 1 5 10 15 TGG TGG TTA CAA TAC TTC CTC ACC AGAGCC GAA GCG CAC TTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Leu Thr Arg AlaGlu Ala His Leu Gln Val 20 25 30 TGG GTC CCC TCT CTC AAC GTT CGA GGA GGCCGC GAT GCC ATC ATC CTC 144 Trp Val Pro Ser Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC ACG TGC GCA GTC TAC CCA GAG CTG ATC TTTGAC ATC ACC AAA CTC 192 Leu Thr Cys Ala Val Tyr Pro Glu Leu Ile Phe AspIle Thr Lys Leu 50 55 60 TTG CTT GCC ACA CTC GGC CCG CTC ATG GTG CTC CAGGCT GGC TTA ACT 240 Leu Leu Ala Thr Leu Gly Pro Leu Met Val Leu Gln AlaGly Leu Thr 65 70 75 80 AGA GTG CCG TAC TTT GTG CGC GCC CAG GGG CTC ATTCGT GCG TGC ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GTT GCT GGG GGC CAT TAT 318 Leu ValArg Lys Val Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:109 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC29, Fig. 16 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 109 CTC TTG ACC TTG TCA CCA TAC TAT AAAGTG TTC CTC GCT AGG CTC ATA 48 Leu Leu Thr Leu Ser Pro Tyr Tyr Lys ValPhe Leu Ala Arg Leu Ile 1 5 10 15 TGG TGG TTG CAA TAT TTT ATC ACC AGAGCC GAA GCG CAC TTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Ile Thr Arg AlaGlu Ala His Leu Gln Val 20 25 30 TGG GTC CCC CCT CTC AAC GTT CGA GGA GGCCGT GAT GCT ATC ATC CTC 144 Trp Val Pro Pro Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC ACG TGC GCA GTC TAC CCA GAG CTA ATC TTTGAC ATC ACC AAA CTC 192 Leu Thr Cys Ala Val Tyr Pro Glu Leu Ile Phe AspIle Thr Lys Leu 50 55 60 TTG CTT GCC ATA CTC GGT CCG CTC ATG GTG CTC CAGGCT AGC ATA ATT 240 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln AlaSer Ile Ile 65 70 75 80 CGA GTG CCG TAC TTC GTA CGC GCT CAA GGC CTC ATTCGT GCA TGC ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GCC GCC GGG GTC AAT TAT 318 Leu ValArg Lys Ala Ala Gly Val Asn Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:110 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC30, Fig. 17 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 110 CTC TTT ACC CTG TCA CCA CAC TGC AAAGTG TTC CTC GCT AGG CTC ATA 48 Leu Phe Thr Leu Ser Pro His Cys Lys ValPhe Leu Ala Arg Leu Ile 1 5 10 15 TGG TGG TTA CAG TAT TTT ATC ACC AGGGCC GAA GCG CAC CTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Ile Thr Arg AlaGlu Ala His Leu Gln Val 20 25 30 TGG ATC CCC CCC CTC AAC GTT CGG GGG GGCCGT GAT GCC ATC ATC CTC 144 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC GCA TGT GCG GTC CAC CCA GAG CTG ATC TTCGAC ATC ACC AAA CTC 192 Leu Ala Cys Ala Val His Pro Glu Leu Ile Phe AspIle Thr Lys Leu 50 55 60 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTG CTC CAGGCT AGC ATA ATT 240 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln AlaSer Ile Ile 65 70 75 80 CGA GTG CCG TAC TTG TAC CGC GCT CAA GGC CTC ATTCGT GCA TGC ATG 288 Arg Val Pro Tyr Leu Tyr Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GCC GCC GGG GGT CAT TAT 318 Leu ValArg Lys Ala Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:111 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC31, Fig. 18 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 111 CTC TTT AAC CTG TCA CCA CAC TAC AAAGTG TTC CTC GCT AGG CTC ATA 48 Leu Phe Asn Leu Ser Pro His Tyr Lys ValPhe Leu Ala Arg Leu Ile 1 5 10 15 TGG TGG TTA CAG TAT TTT ATC ACC AGGGCC GAA GCG CAC CTG CAA GTG 96 Trp Trp Leu Gln Tyr Phe Ile Thr Arg AlaGlu Ala His Leu Gln Val 20 25 30 TGG ATC CCC CCC CTC AAC GTT CAG GGG GGCCGT GAT GCC ATC ATC CTC 144 Trp Ile Pro Pro Leu Asn Val Gln Gly Gly ArgAsp Ala Ile Ile Leu 35 40 45 CTC GCA TGT GCG GTC CAC CCA GAG CTG ATC TTTGAC ATC ACC AAA CTC 192 Leu Ala Cys Ala Val His Pro Glu Leu Ile Phe AspIle Thr Lys Leu 50 55 60 TTG CTC GCC ATA CTC GGT CCG CTC ATG GTG CTC CAGGCT AGC ATA ATT 240 Leu Leu Ala Ile Leu Gly Pro Leu Met Val Leu Gln AlaSer Ile Ile 65 70 75 80 CGA GTG CCG TAC TTC GTA CGC GCT CAA GGC CTC ATTCGT GCA TGC ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GCC GCC GGG GGT CAT TAT 318 Leu ValArg Lys Ala Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:112 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS2-LBC32, Fig. 19 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 112 CTC TTG ACC TTG TCA CCA CAC TAT AAAGTG TTC CTT GCC AGG TTC GTA 48 Leu Leu Thr Leu Ser Pro His Tyr Lys ValPhe Leu Ala Arg Phe Val 1 5 10 15 TGG TGG CTA CAA TAT CTC ATC ACC AGAACC GAA GCG CAT CTG CAA GTG 96 Trp Trp Leu Gln Tyr Leu Ile Thr Arg ThrGlu Ala His Leu Gln Val 20 25 30 TGG GTC CCC CCT CTC AAC GTT CGG GGG GGTCGC GAT GCC ATC ACC CTC 144 Trp Val Pro Pro Leu Asn Val Arg Gly Gly ArgAsp Ala Ile Thr Leu 35 40 45 CTC ACA TGC GTG GTC CAC CCA GAG CTA ATC TTCGAC ATC ACA AAA TAT 192 Leu Thr Cys Val Val His Pro Glu Leu Ile Phe AspIle Thr Lys Tyr 50 55 60 TTG CTC GCC ATA TTC GGC CCG CTC ATG GTG CTC CAGGCC GGC ATA ACT 240 Leu Leu Ala Ile Phe Gly Pro Leu Met Val Leu Gln AlaGly Ile Thr 65 70 75 80 AGA GTG CCG TAC TTC GTG CGC GCA CAA GGG CTC ATTCGT GCA TGC ATG 288 Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile ArgAla Cys Met 85 90 95 TTG GTG CGG AAA GTT GCT GGG GGC CAT TAT 318 Leu ValArg Lys Val Ala Gly Gly His Tyr 100 105 (2) INFORMATION FOR SEQ ID NO:113 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 313 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS5-LBC20, Fig. 20 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 113 C CGT GTT GAG GAG TCA ATT TAC CAATGT TGT GAC TTG GCC CCC GAA 46 Arg Val Glu Glu Ser Ile Tyr Gln Cys CysAsp Leu Ala Pro Glu 1 5 10 15 GCC AAA CTG GCC ATA AAG TCG CCC ACA GAGCGG CTC TAT ATC GGG GGT 94 Ala Lys Leu Ala Ile Lys Ser Pro Thr Glu ArgLeu Tyr Ile Gly Gly 20 25 30 CCC CTG ACT AAT TCA AAA GGG CAG AAC TGC GGTTAC TGC CGG TGC CGC 142 Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys Gly TyrCys Arg Cys Arg 35 40 45 GCG AGC CTG CTG ACG ACT AGC TGC GGT AAT ACC CTCACA TGT CAC CTG 190 Ala Ser Leu Leu Thr Thr Ser Cys Gly Asn Thr Leu ThrCys His Leu 50 55 60 AAA GCC ACT GCG GCC TGT CGA GCT GCG AAG CTC CAG GACTGC ACG ATG 238 Lys Ala Thr Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp CysThr Met 65 70 75 CTC GTG AAC GGA GAC GAC CTT GTC GTT ATC TGT GAA AGC GCGGGG ACC 286 Leu Val Asn Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala GlyThr 80 85 90 95 CAG GAG GAC GCG GCG AGC CTA CGA GTC 313 Gln Glu Asp AlaAla Ser Leu Arg Val 100 (2) INFORMATION FOR SEQ ID NO: 114 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 282 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: NS5-LBC21, Fig. 21 , NS5B-LBC24, Fig. 29(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114 C CGT GTT GAG GAG TCA ATT TACCAA TAT TGT GAC TTG GCC CCC GAA 46 Arg Val Glu Glu Ser Ile Tyr Gln TyrCys Asp Leu Ala Pro Glu 1 5 10 15 GCC AAA CTG GCC ATA AAG TCG CTC ACAGAG CGG CTC TAT ATC GGG GGT 94 Ala Lys Leu Ala Ile Lys Ser Leu Thr GluArg Leu Tyr Ile Gly Gly 20 25 30 CCC CTG ACT AAT TCA AAA GGG CAG AAC TGCGGT TAC CGC CGG TGC CGC 142 Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys GlyTyr Arg Arg Cys Arg 35 40 45 GCG ACC GTG CTG ACG ACT AGC TGC GGT AAT ACCCTC ACA TGT CAC CTG 190 Ala Thr Val Leu Thr Thr Ser Cys Gly Asn Thr LeuThr Cys His Leu 50 55 60 AAA GCC ACT GCG GCC TGT CGA GCT GCG AAA CTC CGGGAC TGC ACG ATG 238 Lys Ala Thr Ala Ala Cys Arg Ala Ala Lys Leu Arg AspCys Thr Met 65 70 75 CTC GTG AAC GGA GAC GAC CTT GTG CTT ATC TGT GAA AGCGCG GG 282 Leu Val Asn Gly Asp Asp Leu Val Leu Ile Cys Glu Ser Ala 80 8590 (2) INFORMATION FOR SEQ ID NO: 115 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 285 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: NS5-LBC23, Fig. 22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115 C CGT GTT GAG GAG TCA ATT TAC CAA TGT TGT GAC TTG GCC CCC GAA 46 ArgVal Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala Pro Glu 1 5 10 15 GCCAAA CTG GCC ATA AAG TCG CTC ACA GAG CGG CTC TAT ATC GGG GGT 94 Ala LysLeu Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly 20 25 30 CCC CTGACT AAT TCA AAA GGG CAG AAC TGC GGT TAC CGC CGG TGC CAC 142 Pro Leu ThrAsn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys His 35 40 45 GCG AGC GGCGTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT CAC 190 Ala Ser Gly ValLeu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys His 50 55 60 CTG AAA GCC ACTGCG GCC TGT CGA GCT GCG AAG CTC CGG GAC TGC ACG 238 Leu Lys Ala Thr AlaAla Cys Arg Ala Ala Lys Leu Arg Asp Cys Thr 65 70 75 ATG CTC GTG AAC GGAGAT GAC CTT GTC GTT ATC TGT GAA AGC GCG GG 285 Met Leu Val Asn Gly AspAsp Leu Val Val Ile Cys Glu Ser Ala 80 85 90 (2) INFORMATION FOR SEQ IDNO: 116 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 282 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: NS5-LBC25, Fig.23 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116 C CGT GTT GAG GAG TCA ATTTAC CAA TGT TGT GAC TTG GCC CCC GAA 46 Arg Val Glu Glu Ser Ile Tyr GlnCys Cys Asp Leu Ala Pro Glu 1 5 10 15 GCC AAA CTG GCC ATA AAG TCG CTCACA GAG CGG CTC TAT ATC GGG GGT 94 Ala Lys Leu Ala Ile Lys Ser Leu ThrGlu Arg Leu Tyr Ile Gly Gly 20 25 30 CCC CTG ACT AAT TCA AAA GGG CAG AACTGC GGT TAC CGC CGG TGC CGC 142 Pro Leu Thr Asn Ser Lys Gly Gln Asn CysGly Tyr Arg Arg Cys Arg 35 40 45 GCG AGC CTG CTG ACG ACT AGC TGC GGT AATACC CTC ACA TGT CAC CTG 190 Ala Ser Leu Leu Thr Thr Ser Cys Gly Asn ThrLeu Thr Cys His Leu 50 55 60 AAA GCC ACT GCG GCC TGT CGA GCT GCG AAG CTCCGG GAC TGC ACG ATG 238 Lys Ala Thr Ala Ala Cys Arg Ala Ala Lys Leu ArgAsp Cys Thr Met 65 70 75 CTC GTG AAC GGA GAC GAC CTT GTC GTT ATC TGT GAAAGC GCG GG 282 Leu Val Asn Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala80 85 90 (2) INFORMATION FOR SEQ ID NO: 117 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 208 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: NS5-LBC27, Fig. 24 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 117 C CGT GTT GAG GAG TCA ATT TAC CAA TGT TGTGAC TTG GCC CCC GAA 46 Arg Val Glu Glu Ser Ile Tyr Gln Cys Cys Asp LeuAla Pro Glu 1 5 10 15 GCC AAA CTG GCC ATA AAG TCG CTC ACA GAG CGG CTCTAT ATC GGG GGT 94 Ala Lys Leu Ala Ile Lys Ser Leu Thr Glu Arg Leu TyrIle Gly Gly 20 25 30 CCC CTG ACT AAT TCA AAA GGG CAG AAC TGC GGT TAC CGCCGG TGC CAC 142 Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg ArgCys His 35 40 45 GCG AGC GGC GTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACATGT CAC 190 Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr CysHis 50 55 60 CTG AAA GCC ACT GCG GCC 208 Leu Lys Ala Thr Ala Ala 65 (2)INFORMATION FOR SEQ ID NO: 118 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:316 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: NS5-LBC28, Fig.25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118C CGT GTT GAG GAG TCA ATT TAC CAA TGT TGT GAC TTG GCC CCC GAA 46 Arg ValGlu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala Pro Glu 1 5 10 15 GCC AAACTG GCC ATA AAG TCG CTC ACA GAG CGG CTC TAT ATC GGG GGT 94 Ala Lys LeuAla Ile Lys Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly 20 25 30 CCC CTG ACTAAT TCA AAA GGG CAG AAC TGC GGT TAC CGC CGG TGC CAC 142 Pro Leu Thr AsnSer Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys His 35 40 45 GCG AGC GGC GTGCTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT CGC 190 Ala Ser Gly Val LeuThr Thr Ser Cys Gly Asn Thr Leu Thr Cys Arg 50 55 60 CTG AAA GCC ACT GCGGCC TGT CGA GCT GCG AAG CTC CGG GAC TGC ACG 238 Leu Lys Ala Thr Ala AlaCys Arg Ala Ala Lys Leu Arg Asp Cys Thr 65 70 75 ATG CTC GTG AAC GGA GATGAC CTT GTC GTT ATC TGT GAA AGC GCG GGG 286 Met Leu Val Asn Gly Asp AspLeu Val Val Ile Cys Glu Ser Ala Gly 80 85 90 95 ACC CAG GAG GAC GCG GCGAGC CTA CGA GTC 316 Thr Gln Glu Asp Ala Ala Ser Leu Arg Val 100 105 (2)INFORMATION FOR SEQ ID NO: 119 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:318 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: NS2-LBC1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119 CTC TTGACC TTG TCA CCA CAC TAT AAA GTG TTC CTT GCC AGG TTC ATA 48 Leu Leu ThrLeu Ser Pro His Tyr Lys Val Phe Leu Ala Arg Phe Ile 1 5 10 15 TGG TGGCTA CAA TAT CTC ATC ACC AGA ACC GAA GCG CAT CTG CAA GTG 96 Trp Trp LeuGln Tyr Leu Ile Thr Arg Thr Glu Ala His Leu Gln Val 20 25 30 TGG GTC CCCCCT CTC AAC GTT CGG GGG GGT CGC GAT GCC ATC ATC CTC 144 Trp Val Pro ProLeu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu 35 40 45 CTC ACA TGC GTGGTC CAC CCA GAG CTA ATC TTT GAC ATC ACA AAA TAT 192 Leu Thr Cys Val ValHis Pro Glu Leu Ile Phe Asp Ile Thr Lys Tyr 50 55 60 TTG CTC GCC ATA TTCGGC CCG CTC ATG GTG CTC CAG GCC GGC ATA ACT 240 Leu Leu Ala Ile Phe GlyPro Leu Met Val Leu Gln Ala Gly Ile Thr 65 70 75 80 AGA GTG CCG TAC TTCGTG CGC GCA CAA GGG CTC ATT CGT GCA TGC ATG 288 Arg Val Pro Tyr Phe ValArg Ala Gln Gly Leu Ile Arg Ala Cys Met 85 90 95 TTG GCG CGG AAA GTC GTGGGG GGT CAT TAC 318 Leu Ala Arg Lys Val Val Gly Gly His Tyr 100 105 (2)INFORMATION FOR SEQ ID NO: 120 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:106 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (ix) FEATURE: (D) OTHER INFORMATION: JHCV-NCI (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 120 Leu Leu Thr Leu Ser Pro Tyr Tyr LysVal Phe Leu Ala Arg Leu 1 5 10 15 Ile Trp Trp Leu Gln Tyr Phe Ile ThrArg Ala Glu Ala His Leu 20 25 30 Gln Val Trp Val Pro Pro Leu Asn Val ArgGly Gly Arg Asp Ala 35 40 45 Ile Ile Leu Leu Thr Cys Ala Val His Pro GluLeu Ile Phe Asp 50 55 60 Ile Thr Lys Leu Leu Leu Ala Ile Leu Gly Pro LeuMet Val Leu 65 70 75 Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg AlaGln Gly 80 85 90 Leu Ile Arg Ala Cys Met Leu Val Arg Lys Val Ala Gly GlyHis 95 100 105 Tyr (2) INFORMATION FOR SEQ ID NO: 121 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 106 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (D) OTHERINFORMATION: JHCV-OSAKA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121 LeuLeu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu 1 5 10 15 IleTrp Trp Leu Gln Tyr Phe Thr Thr Arg Ala Glu Ala Asp Leu 20 25 30 His ValTrp Ile Pro Pro Leu Asn Ala Arg Gly Gly Arg Asp Ala 35 40 45 Ile Ile LeuLeu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp 50 55 60 Ile Thr Lys LeuLeu Ile Ala Ile Leu Gly Pro Leu Met Val Leu 65 70 75 Gln Ala Gly Ile ThrArg Val Pro Tyr Phe Val Arg Ala Gln Gly 80 85 90 Leu Ile His Ala Cys MetLeu Val Arg Lys Val Ala Gly Gly His 95 100 105 Tyr (2) INFORMATION FORSEQ ID NO: 122 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(ix) FEATURE: (D) OTHER INFORMATION: HCPT-CHIRON (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 122 Ala Leu Thr Leu Ser Pro Tyr Tyr Lys Arg TyrIle Ser Trp Cys 1 5 10 15 Leu Trp Trp Leu Gln Tyr Phe Leu Thr Arg ValGlu Ala Gln Leu 20 25 30 His Val Trp Ile Pro Pro Leu Asn Val Arg Gly GlyArg Asp Ala 35 40 45 Val Ile Leu Leu Met Cys Ala Val His Pro Thr Leu ValPhe Asp 50 55 60 Ile Thr Lys Leu Leu Leu Ala Val Phe Gly Pro Leu Trp IleLeu 65 70 75 Gln Ala Ser Leu Leu Lys Val Pro Tyr Phe Val Arg Val Gln Gly80 85 90 Leu Leu Arg Phe Cys Ala Leu Ala Arg Lys Met Ile Gly Gly His 95100 105 Tyr (2) INFORMATION FOR SEQ ID NO: 123 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 316 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: NS5B-LBC1 (xi) SEQUENCE DESCRIPTION: SEQID NO: 123 C CGT GTT GAG GAG TCA ATT TAC CAA TGT TGT GAC TTG GCC CCC GAA46 Arg Val Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala Pro Glu 1 5 10 15GCC AAA CTG GCC ATA AAG TCG CTC ACA GAG CGG CTC TAT ATC GGG GGT 94 AlaLys Leu Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly 20 25 30 CCCCTG ACT AAT TCA AAA GGG CAG AAC TGC GGT TAC CGC CGG TGC CGC 142 Pro LeuThr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg 35 40 45 GCG AGCGGC GTG CTG ACG ACT AGC TGC GGT AAT ACC CTC ACA TGT TAC 190 Ala Ser GlyVal Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr 50 55 60 CTG AAA GCCACT GCG GCC TGT CGA GCT GCG AAG CTC CGG GAC TGC ACG 238 Leu Lys Ala ThrAla Ala Cys Arg Ala Ala Lys Leu Arg Asp Cys Thr 65 70 75 ATG CTC GTG AACGGA GAC GAC CTT GTC GTT ATC TGT GAA AGC GCG GGA 286 Met Leu Val Asn GlyAsp Asp Leu Val Val Ile Cys Glu Ser Ala Gly 80 85 90 95 ACC CAA GAG GATGCG GCG AGC CTA CGA GTC 316 Thr Gln Glu Asp Ala Ala Ser Leu Arg Val 100105 (2) INFORMATION FOR SEQ ID NO: 124 (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 228 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: UBIQUITINE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124 ATGCAA ATT TTC GTC AAA ACT CTA ACA GGG AAG ACT ATA ACC CTA GAG 48 Met GlnIle Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 GTTGAA TCT TCC GAC ACT ATT GAC AAC GTC AAA AGT AAA ATT CAA GAT 96 Val GluSer Ser Asp Thr Ile Asp Asn Val Lys Ser Lys Ile Gln Asp 20 25 30 AAA GAAGGT ATC CCT CCG GAT CAG CAG AGA TTG ATT TTT GCT GGT AAG 144 Lys Glu GlyIle Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 CAA CTA GAAGAT GGT AGA ACC TTG TCT GAC TAC AAC ATC CAA AAG GAA 192 Gln Leu Glu AspGly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 TCT ACT CTT CACTTG GTG TTG AGA CTC CGC GGT GGT 228 Ser Thr Leu His Leu Val Leu Arg LeuArg Gly Gly 65 70 75 (2) INFORMATION FOR SEQ ID NO: 125 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (D) OTHER INFORMATION: primer A (xi) SEQUENCE DESCRIPTION: SEQID NO: 125 CATAGTGGTC TGCGGAACCG 20 (2) INFORMATION FOR SEQ ID NO: 126(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer B (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 126 TTGAGGTTTA GGATTCGTGC 20 (2) INFORMATION FORSEQ ID NO: 127 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHER INFORMATION: primer C(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127 TACACCGGAA TTGCCAGGAC 20 (2)INFORMATION FOR SEQ ID NO: 128 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (D) OTHERINFORMATION: primer D (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128TCATGGTGCA CGGTCTACGA G 21

What is claimed is:
 1. A Korean type hepatitis C virus (KHCV) cDNA whichhas the nucleotide sequence of SEQ ID NO:
 96. 2. A polynucleotideencoding a KHCV polypeptide, selected from the group consisting of: the301st-726th, 301st-855th, 343rd-726th, 343rd-852nd, 343rd-915th,814th-1326th, 916th-1509th, 1201st-2016th, 1510th-2010th, 1945th-2742nd,2011th-2529th, 3208th-3960th, 3475th-3744th, 3916th-4713th,3925th-4563rd, 5422nd-5547th, 6649th-7050th, 7612th-8184th,7642nd-8136th and 8722 nd-9216th nucleotides in the KHCV cDNA of claim1, and a combination thereof.
 3. A recombinant expression vectorcomprising an open reading frame comprising the polynucleotide of claim2, wherein the open reading frame is operably linked to a regulatorysequence compatible with a desired host organism.
 4. The vector of claim3 wherein the open reading frame comprises a polynucleotide encodingubiquitin fused with the polynucleotide of claim
 2. 5. The vector ofclaim 4 which is a yeast expression vector selected from the groupconsisting of pYLBC-A/G-UB-CORE 14(ATCC 74081), pYLBC-A/G-UB-CORE 17,pYLBC-A/G-UB-CORE 22, pYLBC-A/G-UB-KHCV 897, pYLBC-A/G-UB-KHCV 403(ATCC74079), pYLBC-A/G-UB-KHCV 573, pYLBC-A/G-UB-E2N andpYLBC-A/G-UB-E2C(ATCC 74117).
 6. A Saccharomyces sp. cell transformedwith the vector of claim
 5. 7. The vector of claim 4 which is an E. coliexpression vector selected from the group consisting ofptrpH-UB-CORE14(ATCC 68642), ptrpH-UB-CORE17(ATCC 68641),ptrpH-UB-CORE22, ptrpH-UB-KHCV 897(ATCC 68640), ptrpH-UB-E1(ATCC 68878),ptrpH-UB-E2N(ATCC 68966) and ptrpH-UB-E2C.
 8. An E. coli celltransformed with the vector of claim
 7. 9. The vector of claim 4 whichis an E. coli expression vector of pMAL-KHCV 426, pMAL-KHCV 555(ATCC68639), pMAL-KHCV 513, pMAL-KHCV 810, pMAL-KHCV 798, pMAL-KHCV 754,pMAL-KHCV 652, pMAL-KHCV 403, pMAL-KHCV 271, pMAL-KHCV 495 or pMAL-KHCV494.
 10. A recombinant expression vector comprising an open readingframe comprising a polynucleotide encoding a maltose binding proteinfused with the polynucleotide of claim 2, wherein the open reading frameis operably linked to a regulatory sequence compatible with a desiredhost organism.
 11. An E. coli cell transformed with the vector of claim10.
 12. A polypeptide encoded by the polynucleotide of claim
 2. 13. Afused polypeptide wherein the polypeptide of claim 12 is fused withubiquitin or a maltose binding protein.
 14. The polypeptide of claim 13which is produced by a Saccharomyces sp. cell transformed with anexpression vector selected from the group consisting of:pYLBC-A/G-UB-CORE 14(ATCC 74081), pYLBC-A/G-UB-CORE 17,pYLBC-A/G-UB-CORE 22, pYLBC-A/G-UB-KHCV 897, pYLBC-A/G-UB-KHCV 403(ATCC74079), pYLBC-A/G-UB-KHCV 573, pYLBC-A/G-UB-E2N andpYLBC-A/G-UB-E2C(ATCC 74117).
 15. The polypeptide of claim 14 which isselected from the group consisting of: KHCV UB 897 protein, KHCV UB E1protein, KHCV UB 403 protein, KHCV UB CORE 14 protein, KHCV UB 573protein, KHCV UB CORE 17 protein, KHCV UB CORE 22 protein, KHCV UB-E2Nprotein, and KHCV UB-E2C protein.
 16. The polypeptide of claim 13 whichis produced by an E. coli cell transformed with an expression vectorselected from the group consisting of: ptrpH-UB-CORE14(ATCC 68642),ptrpH-UB-CORE17(ATCC 68641), ptrpH-UB-CORE22, ptrpH-UB-KHCV 897(ATCC68640), ptrpH-UB-E1(ATCC 68878), ptrpH-UB-E2N(ATCC 68966) andptrpH-UB-E2C.
 17. The polypeptide of claim 16 which is selected from thegroup consisting of: KHCV UB CORE14, KHCV UB CORE17, KHCV UB CORE22,KHCV UB 897, KHCV UB E1, KHCV UB E2N and KHCV UB E2C.
 18. Thepolypeptide of claim 13 which is produced by an E. coli cell transformedwith a recombinant expression vector comprising an open reading framecontaining a polynucleotide encoding a maltose binding protein fusedwith a KHCV polynucleotide, wherein said open reading frame beingoperably linked to a regulatory sequence compatible with an E. coli celland said KHCV polynucleotide being selected from the group consistingof: the 301st-726th, 301st-855th, 343rd-726th, 343rd-852nd, 343rd-915th,814th-1326th, 916th-1509th, 1201st-2016th, 1510th-2010th, 1945th-2742nd,2011th-2529th, 3208th-3960th, 3475th-3744th, 3916th-4713th,3925th-4563rd, 5422nd-5547th, 6649th-7050th, 7612th-8184th,7642nd-8136th and 8722nd-9216th nucleotides in the KHCV cDNA of SEQ IDNO: 96, and a combination thereof.
 19. The polypeptide of claim 18wherein the expression vector is selected from the group consisting of:pMAL-KHCV 426, pMAL-KHCV 555, pMAL-KHCV 513, pMAL-KHCV 810, pMAL-KHCV798, pMAL-KHCV 754, pMAL-KHCV 652, pMAL-KHCV 403, pMAL-KHCV 271,PMAL-KHCV 495 and pMAL-KHCV
 494. 20. The polypeptide of claim 18 whichis selected from the group consisting of: KHCV 426 protein, KHCV 555protein, KHCV 513 protein, KHCV 810 protein, KHCV 798 protein, KHCV 271protein, KHCV 754 protein, KHCV 652 protein, KHCV 403 protein, KHCV 495protein and KHCV 494 protein.
 21. A diagnostic reagent for detecting anantibody directed against a KHCV antigen in a sample which comprises thepolypeptide of claim 12 as an active ingredient.
 22. A diagnostic kitcomprising the diagnostic reagent of claim
 21. 23. A diagnostic reagentfor detecting an antibody directed against a KHCV antigen in a sample,which comprises two or more polypeptides of claim 12 as an activeingredient.
 24. A monoclonal antibody having an immunoreactivity withthe polypeptide of claim 12, which is useful for the purification anddetection of a KHCV epitope.
 25. The monoclonal antibody of claim 24which is included in an IgG subclass.
 26. The monoclonal antibody ofclaim 24 which is immunoreactive with a KHCV polypeptide encoded by the3916th to 4713th nucleotides in the KHCV cDNA of claim
 1. 27. Theantibody of claim 26 which is immunoreactive with a polypeptide havingthe amino acid sequence of SEQ ID NO:
 94. 28. The antibody of claim 26which is immunoreactive with a polypeptide having the amino acidsequence of SEQ ID NO:
 95. 29. A diagnostic reagent for detecting a KHCVepitope in a sample comprising one or more of the monoclonal antibodiesaccording to any one of claims 24 to 28 as an active ingredient.
 30. Acell line producing a monoclonal antibody having a immunoreactivity witha KHCV epitope, which is produced by the steps of: (a) immunizing ananimal with the polypeptide of claim 12; (b) separating a spleen cellfrom the immunized animal; and (c) fusing the spleen cell with a myelomacell.
 31. The cell line of claim 30 which is Lucky 1.1(ATCC 10949) orLucky 1.2(ATCC 10950).
 32. The polypeptide of claim 12, which isselected from the group consisting of: KHCV 426 protein, KHCV 555protein, KHCV CORE 14 protein, KHCV CORE 17 protein, KHCV CORE 22protein, KHCV 513 protein, KHCV E1 protein, KHCV 810 protein, KHCV E2Nprotein, KHCV 798 protein, KHCV E2C protein, KHCV 271 protein, KHCV 754protein, KHCV 897 protein, KHCV 652 protein, KHCV NS4E protein, KHCV 403protein, KHCV 573 protein, KHCV 495 protein and KHCV 494 protein.
 33. Akit for detecting a polynucleotide derived from KHCV in a samplecomprising an oligonucleotide of 8 or more nucleotides which bindscomplementarily to the KHCV cDNA of SEQ ID NO:
 96. 34. A KHCV cDNAcoding for a KHCV wherein the amino acid sequence encoded in the 2824thto 3141st nucleotide of the KHCV cDNA of SEQ ID NO: 96 is substituted byany one of the amino acid sequences of SEQ ID NO's: 101 to
 112. 35. AKHCV cDNA coding for a KHCV wherein the amino acid sequence encoded inthe 2824th to 3098th nucleotides of the KHCV cDNA of SEQ ID NO: 96 issubstituted by the amino acid sequence of SEQ ID NO:
 97. 36. A kit fordetecting a polynucleotide originating from KHCV in a sample comprisinga nucleotide sequence of 8 or more nucleotides which bindscomplementarily to the KHCV cDNA of claim 34 or 35.